U.S. patent number 11,414,688 [Application Number 16/717,375] was granted by the patent office on 2022-08-16 for processes and systems for preparation of nucleic acid sequencing libraries and libraries prepared using same.
This patent grant is currently assigned to 10X GENOMICS, INC.. The grantee listed for this patent is 10X GENOMICS, INC.. Invention is credited to Zahra Kamila Belhocine, Keith Bjornson, Paul Hardenbol, Benjamin Hindson, Pranav Patel, Indira Wu, Paul William Wyatt.
United States Patent |
11,414,688 |
Hardenbol , et al. |
August 16, 2022 |
Processes and systems for preparation of nucleic acid sequencing
libraries and libraries prepared using same
Abstract
This disclosure provides methods for preparing a sequencing
library including the steps of providing a template nucleic acid
sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP excising
enzyme, and a plurality of beads including oligonucleotide adapter
sequence segments; amplifying the template nucleic acid with the
polymerase, dNTPs, dUTP and random hexamer to provide a
complementary nucleic acid sequence including occasional dUTPs; and
excising the incorporated dUTPs with the dUTP excising enzyme to
provide nicks in the complementary nucleic acid sequence to provide
a sequencing library.
Inventors: |
Hardenbol; Paul (San Francisco,
CA), Patel; Pranav (Fremont, CA), Hindson; Benjamin
(Pleasanton, CA), Wyatt; Paul William (Pleasanton, CA),
Bjornson; Keith (Fremont, CA), Wu; Indira (San Carlos,
CA), Belhocine; Zahra Kamila (Fremont, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
10X GENOMICS, INC. |
Pleasanton |
CA |
US |
|
|
Assignee: |
10X GENOMICS, INC. (Pleasanton,
CA)
|
Family
ID: |
1000006499324 |
Appl.
No.: |
16/717,375 |
Filed: |
December 17, 2019 |
Prior Publication Data
|
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|
|
Document
Identifier |
Publication Date |
|
US 20200190551 A1 |
Jun 18, 2020 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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16228362 |
Dec 20, 2018 |
10557158 |
|
|
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14990276 |
Mar 5, 2019 |
10221436 |
|
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62102420 |
Jan 12, 2015 |
|
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62262769 |
Dec 3, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6806 (20130101); C12P 19/34 (20130101); C40B
20/04 (20130101); C12Q 1/6806 (20130101); C12Q
2525/121 (20130101); C12Q 2525/185 (20130101); C12Q
2525/191 (20130101); C12Q 2525/197 (20130101); C12Q
2531/143 (20130101); C12Q 2535/122 (20130101); C12Q
2563/149 (20130101); B01J 2219/00572 (20130101); B01J
2219/00547 (20130101); B01J 2219/00722 (20130101) |
Current International
Class: |
C40B
20/04 (20060101); C12P 19/34 (20060101); C12Q
1/6806 (20180101) |
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Co-pending U.S. Appl. No. 17/175,542, inventors Maheshwari;
Arundhati Shamoni et al., filed Feb. 12, 2021. cited by applicant
.
Co-pending U.S. Appl. No. 17/220,303, inventor Walter; Dagmar,
filed Apr. 1, 2021. cited by applicant.
|
Primary Examiner: Gross; Christopher M
Attorney, Agent or Firm: Wilson Sonsini Goodrich &
Rosati
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of U.S. application
Ser. No. 16/228,362, filed Dec. 20, 2018, which is a continuation
of U.S. application Ser. No. 14/990,276, filed Jan. 7, 2016, now
U.S. Pat. No. 10,221,436, which claims the benefit of U.S.
Provisional Patent Application No. 62/102,420, filed Jan. 12, 2015
and U.S. Provisional Patent Application No. 62/262,769, filed Dec.
3, 2015, each of which applications are incorporated herein by
reference in their entirety for all purposes.
Claims
What is claimed is:
1. A system, comprising: a reaction mixture comprising a sample
nucleic acid molecule, a plurality of nucleotides, a polymerizing
enzyme, an excising enzyme, a bead comprising a plurality of
nucleic acid barcode molecules, and a primer separate from said
bead, wherein said plurality of nucleotides comprises a
uridine-containing nucleotide, wherein said primer comprises a
random sequence, wherein said polymerizing enzyme is configured to
use said sample nucleic acid molecule and said plurality of
nucleotides, including said uridine-containing nucleotide, to
generate a double-stranded nucleic acid molecule comprising a
uracil, wherein said excising enzyme is configured to excise said
uracil from said double-stranded nucleic acid molecule, and wherein
a nucleic acid barcode molecule of said plurality of nucleic acid
barcode molecules is configured to couple to said double-stranded
nucleic acid molecule or derivative thereof.
2. The system of claim 1, wherein said primer is configured to
anneal to said sample nucleic acid molecule.
3. The system of claim 1, wherein said random sequence of said
primer is 5 to 25 nucleotides in length.
4. The system of claim 1, further comprising a ligating enzyme,
wherein said ligating enzyme is configured to ligate said nucleic
acid barcode molecule to said double-stranded nucleic acid molecule
or derivative thereof.
5. The system of claim 4, wherein said ligating enzyme is a
deoxyribonucleic acid (DNA) ligase.
6. The system of claim 5, wherein said ligating enzyme is a T4 DNA
ligase.
7. The system of claim 1, wherein said nucleic acid barcode
molecule of said plurality of nucleic acid barcode molecules is
double stranded.
8. The system of claim 1, wherein said polymerizing enzyme has
strand displacement activity.
9. The system of claim 8, wherein said polymerizing enzyme is a phi
29 polymerase.
10. The system of claim 1, wherein said excising enzyme is a uracil
DNA glycosylase.
11. The system of claim 1, wherein said bead is a gel bead.
12. The system of claim 11, wherein said gel bead is a degradable
gel bead.
13. The system of claim 1, wherein said plurality of nucleic acid
barcode molecules is releasably coupled to said bead.
14. The system of claim 1, wherein nucleic acid barcode molecules
of said plurality of nucleic acid barcode molecules are
covalently-linked to said bead.
15. The system of claim 1, wherein said uracil is present in only
one strand of said double-stranded nucleic acid molecule.
16. The system of claim 1, wherein said nucleic acid barcode
molecule further comprises one or more functional sequences
selected from the group consisting of an adapter sequence, a primer
sequence, a primer annealing sequence, an attachment sequence, a
sequencing primer sequence, and a partial sequencing primer
sequence.
17. The system of claim 1, further comprising a partition
comprising said reaction mixture.
18. The system of claim 17, wherein said partition is a
droplet.
19. The system of claim 17, wherein said partition is a well.
20. A system, comprising: a reaction mixture comprising a
double-stranded deoxyribonucleic acid (DNA) molecule comprising a
uracil, an excising enzyme configured to excise said uracil from
said double-stranded DNA molecule, a DNA polymerase having strand
displacement activity, and a bead comprising a plurality of nucleic
acid barcode molecules.
21. The system of claim 20, further comprising a ligating enzyme,
wherein said ligating enzyme is configured to ligate a nucleic acid
barcode molecule of said plurality of nucleic acid barcode
molecules to said double-stranded DNA molecule or derivative
thereof.
22. The system of claim 20, wherein said DNA polymerase having
strand displacement activity is configured to generate
single-stranded nucleic acid molecules from said double-stranded
DNA molecule or derivative thereof.
23. The system of claim 20, wherein said excising enzyme is
configured to excise said uracil from said double-stranded DNA
molecule to generate a nicked, double-stranded DNA molecule.
24. The system of claim 20, wherein said uracil is present in only
one strand of said double-stranded DNA molecule.
25. The system of claim 20, wherein said bead is a gel bead.
26. The system of claim 25, wherein said gel bead is a degradable
gel bead.
27. The system of claim 20, wherein said plurality of nucleic acid
barcode molecules is releasably coupled to said bead.
28. The system of claim 20, wherein nucleic acid barcode molecules
of said plurality of nucleic acid barcode molecules are
covalently-linked to said bead.
29. The system of claim 20, wherein a nucleic acid barcode molecule
of said plurality of nucleic acid barcode molecules is configured
to couple to a strand of said double-stranded DNA molecule or
derivative thereof.
30. A system, comprising: a reaction mixture comprising a sample
nucleic acid molecule, a plurality of nucleotides, a DNA
polymerase, an excising enzyme, and a bead comprising a plurality
of nucleic acid barcode molecules, wherein said plurality of
nucleotides comprise a uridine-containing nucleotide, wherein said
DNA polymerase has strand-displacement activity and is configured
to use said sample nucleic acid molecule and said plurality of
nucleotides, including said uridine-containing nucleotide, to
generate a double-stranded nucleic acid molecule comprising a
uracil, wherein said excising enzyme is configured to excise said
uracil from said double-stranded nucleic acid molecule, and wherein
a nucleic acid barcode molecule of said plurality of nucleic acid
barcode molecules is configured to couple to said double-stranded
nucleic acid molecule or derivative thereof.
31. The system of claim 30, further comprising a ligating enzyme,
wherein said ligating enzyme is configured to ligate said nucleic
acid barcode molecule to said double-stranded nucleic acid molecule
or derivative thereof.
32. The system of claim 31, wherein said ligating enzyme is a
deoxyribonucleic acid (DNA) ligase.
33. The system of claim 32, wherein said ligating enzyme is a T4
DNA ligase.
34. The system of claim 30, wherein said nucleic acid barcode
molecule of said plurality of nucleic acid barcode molecules is
double stranded.
35. The system of claim 30, wherein said DNA polymerase is a phi 29
polymerase.
36. The system of claim 30, wherein said excising enzyme is a
uracil DNA glycosylase.
37. The system of claim 30, wherein said bead is a gel bead.
38. The system of claim 37, wherein said gel bead is a degradable
gel bead.
39. The system of claim 30, wherein said plurality of nucleic acid
barcode molecules is releasably coupled to said bead.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created Feb. 7,
2016, is named 43487733201SL.txt and is 1 KB in size.
BACKGROUND
Nucleic acid sequencing technology has experienced rapid and
massive advances over recent years. As compared to gel based
separation methods where nested sets of terminated sequence
extension products were interpreted visually by scientists, today's
sequencing technologies produce enormous amounts of sequence data,
allow illumination of never before sequenced genomes and genome
regions, and provide throughput and costs that allow the widespread
adoption of sequencing into routine biological research and
diagnostics.
Genomic sequencing can be used to obtain information in a wide
variety of biomedical contexts, including diagnostics, prognostics,
biotechnology, and forensic biology. Sequencing may involve basic
methods including Maxam-Gilbert sequencing and chain-termination
methods, or de novo sequencing methods including shotgun sequencing
and bridge PCR, or next-generation methods including polony
sequencing, 454 pyrosequencing, Illumina sequencing, SOLiD
sequencing, Ion Torrent semiconductor sequencing, HeliScope single
molecule sequencing, SMRT.RTM. sequencing, and others. For most
sequencing applications, a sample such as a nucleic acid sample is
processed prior to introduction to a sequencing machine. A sample
may be processed, for example, by amplification or by attaching a
unique identifier. Often unique identifiers are used to identify
the origin of a particular sample.
Despite the huge advances in sequencing technology, or perhaps
illuminated by such huge advances, there exists a need to be able
to create broad, diverse and representative sequencing libraries
from samples of nucleic acids. Further, as the applications of
sequencing technologies expands, the needs for these library
preparation methods to address widely divergent sample types also
increases. For example, the ability to uniformly interrogate the
entire genome, or at least the entire portion of the genome that is
of interest is a significant source of difficulty for molecular
biologists. The lack of uniformity emanates from numerous process
inputs into all of the various sequencing technologies. For
example, fragment size biases may make it more likely that a
sequencing technology will sequence only short fragments of the
genome. Likewise, specific sequence context may increase or
decrease the likelihood that portions of the genome will not be
primed and sequenced, or amplified in pre-sequencing steps, leading
to uneven sequence coverage in the resulting sequence data.
Finally, a host of other characteristics of the sequences, e.g.,
secondary or tertiary structures, or the sequencing technologies,
e.g., long read vs. short read technologies, can lead to biased
representation of the originating sequence within a sequencing
library.
With these challenges, the process of converting sample nucleic
acids into sequenceable libraries has taken on significant
complexity and time commitments, e.g., in fragmentation,
separation, amplification, incorporation of sequencer specific
library components, and clean up. Methods and systems are provided
herein for preparing improved sequencing libraries, as well as the
libraries prepared, that have additional benefits of simplified
workflows.
SUMMARY
Provided are improved methods and systems for preparing libraries
of nucleic acids for use as sequencing libraries, as well as the
libraries prepared using these methods. The libraries described
herein have advantages of improved coverage, low error rates, and
applicability for generation of long range sequence information
from shorter read sequence data.
The present disclosure generally provides methods for the
preparation of sequencing libraries, for example barcode sequencing
libraries, useful, for example, with approaches employing NGS (Next
Generation Sequencing). Sequencing libraries produced as described
herein using a priming free amplification by polymerization at nick
sites (priming free amplification), provide superior sequencing
results, e.g., whole genome sequencing results, when compared to
conventional primer based amplification (primed amplification)
library preparation approaches.
In general in one aspect a method of creating a sequencing library
is provided, including creating a plurality of barcoded nucleic
acid fragments from a template nucleic acid, each of the plurality
of barcoded nucleic acid fragments including a common barcode
sequence; and appending a first adapter sequence to each of the
plurality of barcoded nucleic acid fragments, the first adapter
comprising one or more functional sequences.
In one embodiment the creating step includes contacting the
template nucleic acid with a first set of oligonucleotides, the
first set of oligonucleotides comprising a plurality of barcode
oligonucleotides, each of the plurality of barcode oligonucleotides
having the common barcode sequence and a primer sequence at its 3'
terminus; and annealing the primer sequences on the plurality of
barcode oligonucleotides to the template nucleic acid and extending
the plurality of barcode oligonucleotides along the template
nucleic acid to create the plurality of barcoded nucleic acid
fragments from the template nucleic acid.
In another embodiment the appending step includes contacting the
plurality of barcoded nucleic acid fragments with a second set of
oligonucleotides, the second set of oligonucleotides comprising a
plurality of primer sequences complementary to at least a portion
of the plurality of barcoded nucleic acid fragments, and at least
one functional sequence; and annealing the second set of
oligonucleotides to the plurality of barcoded nucleic acid
fragments and extending the second set of oligonucleotides along
the plurality of barcoded nucleic acid fragments, to create
replicate barcoded fragments including the at least one functional
sequence.
In yet another embodiment the appending step includes ligating the
first adapter sequence to each of the plurality of barcoded nucleic
acid fragments. It is envisioned that the step of ligating the
first adapter sequence to each of the plurality of barcoded nucleic
acid fragments includes shearing each of the plurality of barcoded
nucleic acid fragments to create sheared fragments and ligating the
first adapter sequence to a 3' terminus of the sheared
fragments.
In general, in one aspect a method of preparing a sequencing
library is provided including the steps of: (a) providing a
template nucleic acid sequence, dNTPs, dUTP, a primer, a
polymerase, a dUTP excising enzyme, and a plurality of beads
including oligonucleotide adapter sequence segments; (b) amplifying
the template nucleic acid with the polymerase, dNTPs, dUTP and
random hexamer to provide a complementary nucleic acid sequence
including occasional dUTPs; and (c) excising the incorporated dUTPs
with the dUTP excising enzyme to provide nicks in the complementary
nucleic acid sequence to provide a sequencing library.
In one embodiment the method further includes a step (d) of
amplifying the nicked complementary nucleic acid sequence, and a
step (e) of extending the sequence of the amplified nucleic acid
sequence using a nucleic acid extension means. In some embodiments
the steps of the method above are performed in a single
reaction.
In another embodiment the plurality of beads is a pooled bead
population. In a specific embodiment the beads of the pooled bead
population are co-partitioned with one or more of the components
listed in step (a), and wherein the partition optionally comprises
a droplet in an emulsion.
In some embodiments the beads including degradable beads selected
from chemically degradable beads, photodegradable beads and
thermally degradable beads. In a specific embodiment the beads
include chemically reducible cross-linkers. More specifically the
chemically reducible cross-linkers can include disulfide
linkages.
In another embodiment the amplification in step (b) is
isothermal.
In a further embodiment the polymerase is phi29 DNA polymerase.
In a different embodiment the nucleic acid extension means is
selected from the group consisting of a ligating enzyme, a nucleic
acid extension enzyme and a transposase. In a related embodiment
the library of amplified nucleic acid sequences includes single
stranded DNA and the ligating enzyme includes an ATP independent
enzyme. The ATP independent enzyme can include thermostable 5' App
DNA/RNA ligase. In another related embodiment the ligating enzyme
includes a topoisomerase. Specifically the topoisomerase can be
topoisomerase I. In still another related embodiment the ligating
enzyme includes T4 DNA ligase.
In general, in another aspect a method of preparing a barcode
sequencing library is provided, including: (a) providing a template
nucleic acid sequence, dNTPs, dUTP, a primer, a polymerase, a dUTP
excising enzyme, a nucleic acid extension means and a plurality of
beads comprising oligonucleotide barcode sequence segments; (b)
amplifying the template nucleic acid with the polymerase, dNTPs,
dUTP and random hexamer to provide a complementary nucleic acid
sequence including occasional dUTPs; and (c) excising the
incorporated dUTPs with the dUTP excising enzyme to provide nicks
in the complementary nucleic acid sequence; (d) amplifying the
nicked complementary nucleic acid sequence to provide a library of
amplified nucleic acid sequences; and (e) releasing the barcode
sequence segments from the pooled bead population; and (f)
extending the sequence of the amplified nucleic acid sequences
using the barcode sequence segments and the nucleic acid extension
means to provide a barcode library or alternatively, ligating the
barcode sequence segments, using a nucleic acid ligating enzyme, to
the library of amplified nucleic acid sequences to provide a
barcode library.
In some embodiments of the method, the steps are performed in a
single reaction. In one embodiment the plurality of beads is a
pooled bead population. In another embodiment the beads of the
pooled bead population are co-partitioned with one or more of the
components listed in step (a), and wherein the partition optionally
includes a droplet in an emulsion. In a further embodiment the
beads include degradable beads selected from chemically degradable
beads, photodegradable beads and thermally degradable beads. In a
particular embodiment the beads include chemically reducible
cross-linkers. The chemically reducible cross-linkers can include
disulfide linkages.
In other embodiments the amplification in step (b) is isothermal.
In some embodiments the polymerase is phi29 DNA polymerase. In
other embodiments the nucleic acid extension means is selected from
the group consisting of a ligating enzyme, a nucleic acid extension
enzyme and a transposase. In some embodiments the library of
amplified nucleic acid sequences includes single stranded DNA and
the ligating enzyme includes an ATP independent enzyme. In a
specific embodiment the ATP independent enzyme includes
thermostable 5' App DNA/RNA ligase. In a different embodiment the
ligating enzyme includes a topoisomerase. It is contemplated that
the topoisomerase can be topoisomerase I.
In yet another embodiment the ligating enzyme includes T4 DNA
ligase.
In one embodiment the barcode sequence segments include at least 4
nucleotides at least 10 nucleotides or at least 20 nucleotides. In
another embodiment the barcode sequence segments include at least
1000 different barcode sequence segments. In some embodiments at
least 1,000,000 oligonucleotide molecules are attached to each
bead. In other embodiments the pooled bead population includes at
least 10 different bead populations. In a different embodiment the
pooled bead population includes at least 100 different bead
populations. In one specific embodiment the pooled bead population
includes at least 500 different bead populations.
In a further embodiment the oligonucleotide barcode sequence
segments include at least one functional sequence. In one
embodiment the functional sequence is selected from an adapter, a
primer sequence, a primer annealing sequence, an attachment
sequence, and a sequencing primer sequence. In a particular
embodiment the functional sequence is sequestered and releasable in
a releasing step including a stimulus selected from the list
consisting of thermal increase and chemical cleavage. In a
different embodiment the releasing step includes degrading at least
a portion the beads of the bead population including
oligonucleotide barcode sequence segments. In a specific embodiment
degrading the beads includes cleaving a chemical linkage including
a disulfide bridge linkage between the barcode sequence segments
and the bead, and the releasing step includes exposing the beads to
a reducing agent. In a particular embodiment the reducing agent
includes a reducing agent selected from the group consisting of DTT
and TCEP.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the process of priming free
amplification of templates.
FIG. 2A is a diagram illustrating barcoding of templates using an
extension barcoding approach.
FIG. 2B is a diagram illustrating barcoding of single or double
stranded templates using a ligation approach.
FIG. 2C is a diagram illustrating barcoding of single stranded
library molecules using and APP DNA/RNA ligase approach.
FIG. 3 shows results of testing for T base bias based on whole
genome sequencing data.
FIG. 4A is a plot of a primed amplification showing coverage
evenness over 1000 base pairs binned GC content of the human
genome.
FIG. 4B is a plot of a primer free amplification showing coverage
evenness over 1000 base pairs binned GC content of the human
genome.
FIG. 5A is a GC coverage plot for a reaction with no dUTP
added.
FIG. 5B is a GC coverage plot for a reaction with 0.5% dUTP
added.
FIG. 5C is a GC coverage plot for a reaction with 1% dUTP
added.
FIG. 5D is a GC coverage plot for a reaction with 2% dUTP
added.
FIG. 5E is a GC coverage plot for a reaction with 3% dUTP
added.
FIG. 6 shows the results of titration of dUTP and the effect on
chimera rate, Depth Positional CV (DPCV).
FIG. 7 shows the results of DTT addition on DPCV and amplification
rate.
FIG. 8A shows the effect of SSB, DTT or both on DPCV using standard
conditions.
FIG. 8B shows the effect of addition of SSB on amplification
rate.
FIG. 8C shows the effect of addition of SSB on chimera
reduction.
FIG. 9 shows the effect of time on DPCV and amplification
rates.
FIG. 10 shows DPCV and amplification rates with and without
denaturation steps.
FIG. 11 shows the effect of adaptor concentration on dup rate
(measure of library complexity), and DPCV.
FIG. 12A shows the effect of barcoding ligation reaction time on
DPCV.
FIG. 12B shows the effect of barcoding ligation reaction time on
insert size.
FIG. 12C shows the effect of barcoding ligation reaction time on
chimeras.
FIG. 12D shows the effect of barcoding ligation reaction time on
unmapped fraction.
FIG. 12E shows the effect of barcoding ligation reaction time on
amplification rate.
FIG. 13 shows the results of control experiments to test the
specificity of T4 ligase based barcoding.
FIG. 14A is a histogram illustrating evenness of sequencing
coverage in a primed amplification reaction.
FIG. 14B is a histogram illustrating evenness of sequencing
coverage in a primer free amplification.
FIG. 15 shows the effect of nMer concentration (uM) on five
different barcoded template library samples.
FIG. 16 shows the effect of SPRI (Solid Phase Reversible
Immobilization) stringency cut on six different barcoded template
library samples.
FIG. 17 shows the effect of total reaction time on DPCV on five
different barcoded template library samples.
FIG. 18 shows the effect of Uracil-Specific Excision Reagent
(USER.RTM.) concentration on DPCV for six different barcoded
template library samples.
FIG. 19A schematically illustrates an overview of a process for
preparation of barcoded sequencing libraries. FIGS. 19B-19F
schematically illustrate steps of a process for preparation of
barcoded sequencing libraries.
FIG. 20A, FIG. 20B and FIG. 20C schematically illustrate
alternative processes for preparing barcoded sequencing
libraries.
FIG. 21 illustrates a comparison of different enzyme performances
in preparing sequencing libraries.
FIG. 22 schematically illustrates processing of barcoded fragments
of nucleic acids in preparation of sequencing libraries.
FIG. 23 schematically illustrates alternative processes for further
processing fragment nucleic acids in the preparation of sequencing
libraries.
FIG. 24 schematically illustrates an alternative library generation
process.
FIG. 25 schematically illustrates a library barcoding process
utilizing ligation processes in place of primer extension
processes.
DETAILED DESCRIPTION
I. General Overview
Library Preparation Using Priming Free Amplification by
Polymerization at Nick Sites
Sequencing libraries produced as described herein using a priming
free amplification by polymerization at nick sites (priming free
amplification), provide superior sequencing results, e.g., whole
genome sequencing results, when compared to conventional primer
based amplification (primed amplification) library preparation
approaches. Advantageously, for example, the priming free
amplification approach results in more even sequencing coverage
across a broad range of GC base content when compared to primed
amplification results. Additionally, an improved sequencing
coverage evenness is achieved in priming free amplification,
resulting in a more poissonian distribution when compared to the
distribution for primped amplification.
The design of the invention generally is shown in FIG. 1, which
illustrates the process of library preparation using priming free
amplification of templates. The approach illustrated is also
employed in the experimental or prophetic exemplary support as
disclosed in the Examples below. In some embodiments, the
sequencing libraries are tagged with molecular barcodes and are
suitable for use in NGS (Next Generation Sequencing) reactions.
Although illustrated as a series of panels in FIG. 1, the reaction
processes illustrated can be performed simultaneously with all the
reagents present together in the priming free amplification by
polymerization process. This process can be contrasted with a
standard primed amplification process for preparing a sequencing
library.
In general, one method of the invention is shown in FIG. 1. At FIG.
1 (101), a DNA polymerase, for example, phi29 DNA Polymerase (New
England Biolabs.RTM. Inc. (NEB), Ipswich, Mass.) used to perform
isothermal amplification is shown including: initiation using a
hexamer (short arrow) and phi29 DNA polymerase (oval) which has
very high processivity and fidelity that results in even coverage
and low error rates. As the polymerase processes along the target
sequence (long line) a copied DNA template is produced. FIG. 1
(102) illustrates the polymerase based incorporation of dUTP (U) in
a growing template strand (long arrow) upon initial amplification
in the presence of all dNTPs and a small amount of dUTP. FIG. 1
(103) shows the inclusion in the reaction of an enzyme (oval with
bolt) capable of excising dUTP and creating nicks in the copied
template DNA strand (long arrow), but not the original target
sequence (long line). FIG. 1 (104) shows the result of nicking by
the enzyme capable of excising dUTP wherein the original amplified
strand from (103) is now, for example, four shorter amplified
strands (short arrows). Additionally, phi29 DNA polymerase (oval)
is shown engaging at the nick sites for additional amplification in
a priming independent amplification process. FIG. 1 (105)
illustrates recycling of the original target sequence as a template
(long line) upon strand displacement of released amplified
fragments (short arrows) owing to the highly processive phi29 DNA
polymerase (oval). Subsequent amplifications mirror the process
shown in (102) to produce additional released amplified fragments
(short arrows).
This disclosure provides methods, systems and compositions useful
in the processing of sample materials through the controlled
delivery of reagents to subsets of sample components, followed by
analysis of those sample components employing, in part, the
delivered reagents. In many cases, the methods and compositions are
employed for sample processing, particularly for nucleic acid
analysis applications, generally, and nucleic acid sequencing
applications, in particular. Included within this disclosure are
bead compositions that include diverse sets of reagents, such as
diverse libraries of beads attached to large numbers of
oligonucleotides containing barcode sequences, and methods of
making and using the same.
Methods of making beads can generally include, e.g. combining bead
precursors (such as monomers or polymers), primers, and
cross-linkers in an aqueous solution, combining said aqueous
solution with an oil phase, sometimes using a microfluidic device
or droplet generator, and causing water-in-oil droplets to form. In
some cases, a catalyst, such as an accelerator and/or an initiator,
may be added before or after droplet formation. In some cases,
initiation may be achieved by the addition of energy, such, as for
example via the addition of heat or light (e.g., UV light). A
polymerization reaction in the droplet can occur to generate a
bead, in some cases covalently linked to one or more copies of an
oligonucleotide (e.g., primer). Additional sequences can be
attached to the functionalized beads using a variety of methods. In
some cases, the functionalized beads are combined with a template
oligonucleotide (e.g., containing a barcode) and partitioned such
that on average one or fewer template oligonucleotides occupy the
same partition as a functionalized bead. While the partitions may
be any of a variety of different types of partitions, e.g., wells,
microwells, tubes, vials, microcapsules, etc., in preferred
aspects, the partitions may be droplets (e.g., aqueous droplets)
within an emulsion. The oligonucleotide (e.g., barcode) sequences
can be attached to the beads within the partition by a reaction
such as a primer extension reaction, ligation reaction, or other
methods. For example, in some cases, beads functionalized with
primers are combined with template barcode oligonucleotides that
comprise a binding site for the primer, enabling the primer to be
extended on the bead. After multiple rounds of amplification,
copies of the single barcode sequence are attached to the multiple
primers attached to the bead. After attachment of the barcode
sequences to the beads, the emulsion can be broken and the barcoded
beads (or beads linked to another type of amplified product) can be
separated from beads without amplified barcodes. Additional
sequences, such as a random sequence (e.g., a random N-mer) or a
targeted sequence, can then be added to the bead-bound barcode
sequences, using, for example, primer extension methods or other
amplification reactions. This process can generate a large and
diverse library of barcoded beads.
Functional sequences are envisioned to include, for example,
immobilization sequences for immobilizing barcode containing
sequences onto surfaces, e.g., for sequencing applications. For
ease of discussion, a number of specific functional sequences are
described below, such as P5, P7, R1, R2, sample indexes, random
Nmers, etc., and partial sequences for these, as well as
complements of any of the foregoing. However, it will be
appreciated that these descriptions are for purposes of discussion,
and any of the various functional sequences included within the
barcode containing oligonucleotides may be substituted for these
specific sequences, including without limitation, different
attachment sequences, different sequencing primer regions,
different n-mer regions (targeted and random), as well as sequences
having different functions, e.g., secondary structure forming,
e.g., hairpins or other structures, probe sequences, e.g., to allow
interrogation of the presence or absence of the oligonucleotides or
to allow pull down of resulting amplicons, or any of a variety of
other functional sequences.
Also included within this disclosure are methods of sample
preparation for nucleic acid analysis, and particularly for
sequencing applications. Sample preparation can generally include,
e.g. obtaining a sample comprising sample nucleic acid from a
source, optionally further processing the sample, combining the
sample nucleic acid with barcoded beads, and forming emulsions
containing fluidic droplets comprising the sample nucleic acid and
the barcoded beads. Droplets may be generated, for example, with
the aid of a microfluidic device and/or via any suitable
emulsification method. The fluidic droplets can also comprise
agents capable of dissolving, degrading, or otherwise disrupting
the barcoded beads, and/or disrupting the linkage to attached
sequences, thereby releasing the attached barcode sequences from
the bead. The barcode sequences may be released either by degrading
the bead, detaching the oligonucleotides from the bead such as by a
cleavage reaction, or a combination of both. By amplifying (e.g.,
via amplification methods described herein) the sample nucleic acid
in the fluidic droplets, for example, the free barcode sequences
can be attached to the sample nucleic acid. The emulsion comprising
the fluidic droplets can then be broken and, if desired, additional
sequences (e.g., sequences that aid in particular sequencing
methods, additional barcode sequences, etc.) can then be added to
the barcoded sample nucleic acid using, for example, additional
amplification methods. Sequencing can then be performed on the
barcoded, amplified sample nucleic acid and one or more sequencing
algorithms applied to interpret the sequencing data. As used
herein, the sample nucleic acids may include any of a wide variety
of nucleic acids, including, e.g., DNA and RNA, and specifically
including for example, genomic DNA, cDNA, mRNA total RNA, and cDNA
created from a mRNA or total RNA transcript.
The methods and compositions of this disclosure may be used with
any suitable digital processor. The digital processor may be
programmed, for example, to operate any component of a device
and/or execute methods described herein. In some embodiments, bead
formation may be executed with the aid of a digital processor in
communication with a droplet generator. The digital processor may
control the speed at which droplets are formed or control the total
number of droplets that are generated. In some embodiments,
attaching barcode sequences to sample nucleic acid may be completed
with the aid of a microfluidic device and a digital processor in
communication with the microfluidic device. In some cases, the
digital processor may control the amount of sample and/or beads
provided to the channels of the microfluidic device, the flow rates
of materials within the channels, and the rate at which droplets
comprising barcode sequences and sample nucleic acid are
generated.
The methods and compositions of this disclosure may be useful for a
variety of different molecular biology applications including, but
not limited to, nucleic acid sequencing, protein sequencing,
nucleic acid quantification, sequencing optimization, detecting
gene expression, quantifying gene expression, epigenetic
applications, and single-cell analysis of genomic or expressed
markers. Moreover, the methods and compositions of this disclosure
have numerous medical applications including identification,
detection, diagnosis, treatment, staging of, or risk prediction of
various genetic and non-genetic diseases and disorders including
cancer.
II. Beads or Particles
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable bead or particle, including gel beads and
other types of beads. Beads may serve as a carrier for reagents
that are to be delivered in accordance with the methods described
herein. In particular, these beads may provide a surface to which
reagents are releasably attached, or a volume in which reagents are
entrained or otherwise releasably partitioned. These reagents may
then be delivered in accordance with a desired method, for example,
in the controlled delivery of reagents into discrete partitions. A
wide variety of different reagents or reagent types may be
associated with the beads, where one may desire to deliver such
reagents to a partition. Non-limiting examples of such reagents
include, e.g., enzymes, polypeptides, antibodies or antibody
fragments, labeling reagents, e.g., dyes, fluorophores,
chromophores, etc., nucleic acids, polynucleotides,
oligonucleotides, and any combination of two or more of the
foregoing. In some cases, the beads may provide a surface upon
which to synthesize or attach oligonucleotide sequences. Various
entities including oligonucleotides, barcode sequences, primers,
crosslinkers and the like may be associated with the outer surface
of a bead. In the case of porous beads, an entity may be associated
with both the outer and inner surfaces of a bead. The entities may
be attached directly to the surface of a bead (e.g., via a covalent
bond, ionic bond, van der Waals interactions, etc.), may be
attached to other oligonucleotide sequences attached to the surface
of a bead (e.g. adaptor or primers), may be diffused throughout the
interior of a bead and/or may be combined with a bead in a
partition (e.g. fluidic droplet). In preferred embodiments, the
oligonucleotides are covalently attached to sites within the
polymeric matrix of the bead and are therefore present within the
interior and exterior of the bead. In some cases, an entity such as
a cell or nucleic acid is encapsulated within a bead. Other
entities including amplification reagents (e.g., PCR reagents,
primers) may also be diffused throughout the bead or
chemically-linked within the interior (e.g., via pores, covalent
attachment to polymeric matrix) of a bead.
Beads may serve to localize entities or samples. In some
embodiments, entities (e.g. oligonucleotides, barcode sequences,
primers, crosslinkers, adaptors and the like) may be associated
with the outer and/or an inner surface of the bead. In some cases,
entities may be located throughout the bead. In some cases, the
entities may be associated with the entire surface of a bead or
with at least half the surface of the bead.
Beads may serve as a support on which to synthesize oligonucleotide
sequences. In some embodiments, synthesis of an oligonucleotide may
comprise a ligation step. In some cases, synthesis of an
oligonucleotide may comprise ligating two smaller oligonucleotides
together. In some cases, a primer extension or other amplification
reaction may be used to synthesize an oligonucleotide on a bead via
a primer attached to the bead. In such cases, a primer attached to
the bead may hybridize to a primer binding site of an
oligonucleotide that also contains a template nucleotide sequence.
The primer can then be extended by a primer extension reaction or
other amplification reaction, and an oligonucleotide complementary
to the template oligonucleotide can thereby be attached to the
bead. In some cases, a set of identical oligonucleotides associated
with a bead may be ligated to a set of diverse oligonucleotides,
such that each identical oligonucleotide is attached to a different
member of the diverse set of oligonucleotides. In other cases, a
set of diverse oligonucleotides associated with a bead may be
ligated to a set of identical oligonucleotides.
Bead Characteristics
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable bead. In some embodiments, a bead may be
porous, non-porous, solid, semi-solid, semi-fluidic, or fluidic. In
some embodiments, a bead may be dissolvable, disruptable, or
degradable. In some cases, a bead may not be degradable. In some
embodiments, the bead may be a gel bead. A gel bead may be a
hydrogel bead. A gel bead may be formed from molecular precursors,
such as a polymeric or monomeric species. A semi-solid bead may be
a liposomal bead. Solid beads may comprise metals including iron
oxide, gold, and silver. In some cases, the beads are silica beads.
In some cases, the beads are rigid. In some cases, the beads may be
flexible.
In some embodiments, the bead may contain molecular precursors
(e.g., monomers or polymers), which may form a polymer network via
polymerization of the precursors. In some cases, a precursor may be
an already polymerized species capable of undergoing further
polymerization via, for example, a chemical cross-linkage. In some
cases, a precursor comprises one or more of an acrylamide or a
methacrylamide monomer, oligomer, or polymer. In some cases, the
bead may comprise prepolymers, which are oligomers capable of
further polymerization. For example, polyurethane beads may be
prepared using prepolymers. In some cases, the bead may contain
individual polymers that may be further polymerized together. In
some cases, beads may be generated via polymerization of different
precursors, such that they comprise mixed polymers, co-polymers,
and/or block co-polymers.
A bead may comprise natural and/or synthetic materials, including
natural and synthetic polymers. Examples of natural polymers
include proteins and sugars such as deoxyribonucleic acid, rubber,
cellulose, starch (e.g. amylose, amylopectin), proteins, enzymes,
polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran,
collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac,
sterculia gum, xanthan gum, Corn sugar gum, guar gum, gum karaya,
agarose, alginic acid, alginate, or natural polymers thereof.
Examples of synthetic polymers include acrylics, nylons, silicones,
spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,
polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,
polylactic acid, silica, polystyrene, polyacrylonitrile,
polybutadiene, polycarbonate, polyethylene, polyethylene
terephthalate, poly (chlorotrifluoroethylene), poly(ethylene
oxide), poly (ethylene terephthalate), polyethylene,
polyisobutylene, poly(methyl methacrylate), poly(oxymethylene),
polyformaldehyde, polypropylene, polystyrene,
poly(tetrafluoroethylene), poly(vinyl), poly(vinyl alcohol),
poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene
diflu acetate oride materials), poly(vinyl fluoride) and
combinations (e.g., co-polymers) thereof Beads may also be formed
from other than polymers, including lipids, micelles, ceramics,
glass-ceramics, material composites, metals, other inorganic
materials, and others.
In some cases, a chemical cross-linker may be a precursor used to
cross-link monomers during polymerization of the monomers and/or
may be used to functionalize a bead with a species. In some cases,
polymers may be further polymerized with a cross-linker species or
other type of monomer to generate a further polymeric network.
Non-limiting examples of chemical cross-linkers (also referred to
as a "crosslinker" or a "crosslinker agent" herein) include
cystamine, gluteraldehyde, dimethyl suberimidate,
N-Hydroxysuccinimide crosslinker B S3, formaldehyde, carbodiimide
(EDC), SMCC, Sulfo-SMCC, vinylsilance, N,N'diallyltartardiamide
(DATD), N,N'-Bis(acryloyl)cystamine (BAC), or homologs thereof In
some cases, the crosslinker used in the present disclosure contains
cystamine.
Crosslinking may be permanent or reversible, depending upon the
particular crosslinker used. Reversible crosslinking may allow for
the polymer to linearize or dissociate under appropriate
conditions. In some cases, reversible cross-linking may also allow
for reversible attachment of a material bound to the surface of a
bead. In some cases, a cross-linker may form disulfide linkages. In
some cases, the chemical cross-linker forming disulfide linkages
may be cystamine or a modified cystamine. In some embodiments,
disulfide linkages may be formed between molecular precursor units
(e.g. monomers, oligomers, or linear polymers). In some
embodiments, disulfide linkages may be may be formed between
molecular precursor units (e.g. monomers, oligomers, or linear
polymers) or precursors incorporated into a bead and
oligonucleotides.
Cystamine (including modified cystamines), for example, is an
organic agent comprising a disulfide bond that may be used as a
crosslinker agent between individual monomeric or polymeric
precursors of a bead. Polyacrylamide may be polymerized in the
presence of cystamine or a species comprising cystamine (e.g., a
modified cystamine) to generate polyacrylamide gel beads comprising
disulfide linkages (e.g., chemically degradable beads comprising
chemically-reducible cross-linkers). The disulfide linkages may
permit the bead to be degraded (or dissolved) upon exposure of the
bead to a reducing agent.
In at least one alternative example, chitosan, a linear
polysaccharide polymer, may be crosslinked with glutaraldehyde via
hydrophilic chains to form a bead. Crosslinking of chitosan
polymers may be achieved by chemical reactions that are initiated
by heat, pressure, change in pH, and/or radiation.
In some embodiments, the bead may comprise covalent or ionic bonds
between polymeric precursors (e.g. monomers, oligomers, linear
polymers), oligonucleotides, primers, and other entities. In some
cases, the covalent bonds comprise carbon-carbon bonds or thioether
bonds.
In some cases, a bead may comprise an acrydite moiety, which in
certain aspects may be used to attach one or more species (e.g.,
barcode sequence, primer, other oligonucleotide) to the bead. In
some cases, an acrydite moiety can refer to an acrydite analogue
generated from the reaction of acrydite with one or more species,
such as, for example, the reaction of acrydite with other monomers
and cross-linkers during a polymerization reaction. Acrydite
moieties may be modified to form chemical bonds with a species to
be attached, such as an oligonucleotide (e.g., barcode sequence,
primer, other oligonucleotide). For example, acrydite moieties may
be modified with thiol groups capable of forming a, disulfide bond
or may be modified with groups already comprising a disulfide bond.
The thiol or disulfide (via disulfide exchange) may be used as an
anchor point for a species to be attached or another part of the
acrydite moiety may be used for attachment. In some cases,
attachment is reversible, such that when the disulfide bond is
broken (e.g., in the presence of a reducing agent), the agent is
released from the bead. In other cases, an acrydite moiety
comprises a reactive hydroxyl group that may be used for
attachment.
Functionalization of beads for attachment of other species, e.g.,
nucleic acids, may be achieved through a wide range of different
approaches, including activation of chemical groups within a
polymer, incorporation of active or activatable functional groups
in the polymer structure, or attachment at the pre-polymer or
monomer stage in bead production.
For example, in some examples, precursors (e.g., monomers,
cross-linkers) that are polymerized to form a bead may comprise
acrydite moieties, such that when a bead is generated, the bead
also comprises acrydite moieties. Often, the acrydite moieties are
attached to an oligonucleotide sequence, such as a primer (e.g., a
primer for one or more of amplifying target nucleic acids and/or
sequencing target nucleic acids barcode sequence, binding sequence,
or the like)) that is desired to be incorporated into the bead. In
some cases, the primer comprises a P5 sequence. For example,
acrylamide precursors (e.g., cross-linkers, monomers) may comprise
acrydite moieties such that when they are polymerized to form a
bead, the bead also comprises acrydite moieties.
In some cases, precursors such as monomers and cross-linkers may
comprise, for example, a single oligonucleotide (e.g., such as a
primer or other sequence) or other species. In some cases,
precursors such as monomers and cross-linkers may comprise multiple
oligonucleotides, other sequences, or other species. The inclusion
of multiple acrydite moieties or other linker species in each
precursor may improve loading of a linked species (e.g., an
oligonucleotide) into beads generated from the precursors because
each precursor can comprise multiple copies of a species to be
loaded.
In some cases, precursors comprising a functional group that is
reactive or capable of being activated such that it becomes
reactive can be polymerized with other precursors to generate gel
beads comprising the activated or activatable functional group. The
functional group may then be used to attach additional species
(e.g., disulfide linkers, primers, other oligonucleotides, etc.) to
the gel beads. For example, some precursors comprising a carboxylic
acid (COOH) group can co-polymerize with other precursors to form a
gel bead that also comprises a COOH functional group. In some
cases, acrylic acid (a species comprising free COOH groups),
acrylamide, and bis(acryloyl)cystamine can be co-polymerized
together to generate a gel bead comprising free COOH groups. The
COOH groups of the gel bead can be activated (e.g., via
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
N-Hydroxysuccinimide (NETS) or
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)) such that they are reactive (e.g., reactive to amine
functional groups where EDC/NHS or DMTMM are used for activation).
The activated COOH groups can then react with an appropriate
species (e.g., a species comprising an amine functional group where
the carboxylic acid groups are activated to be reactive with an
amine functional group) comprising a moiety to be linked to the
bead.
Beads comprising disulfide linkages in their polymeric network may
be functionalized with additional species via reduction of some of
the disulfide linkages to free thiols. The disulfide linkages may
be reduced via, for example, the action of a reducing agent (e.g.,
DTT, TCEP, etc.) to generate free thiol groups, without dissolution
of the bead. Free thiols of the beads can then react with free
thiols of a species or a species comprising another disulfide bond
(e.g., via thiol-disulfide exchange)) such that the species can be
linked to the beads (e.g., via a generated disulfide bond). In some
cases, though, free thiols of the beads may react with any other
suitable group. For example, free thiols of the beads may react
with species comprising an acrydite moiety. The free thiol groups
of the beads can react with the acrydite via Michael addition
chemistry, such that the species comprising the acrydite is linked
to the bead. In some cases, uncontrolled reactions can be prevented
by inclusion of a thiol capping agent such as, for example,
N-ethylmalieamide or iodoacetate.
Activation of disulfide linkages within a bead can be controlled
such that only a small number of disulfide linkages are activated.
Control may be exerted, for example, by controlling the
concentration of a reducing agent used to generate free thiol
groups and/or concentration of reagents used to form disulfide
bonds in bead polymerization. In some cases, a low concentration
(e.g., molecules of reducing agent:gel bead ratios of less than
about 10000, 100000, 1000000, 10000000, 100000000, 1000000000,
10000000000, or 100000000000) of reducing agent may be used for
reduction. Controlling the number of disulfide linkages that are
reduced to free thiols may be useful in ensuring bead structural
integrity during functionalization. In some cases, optically-active
agents, such as fluorescent dyes may be may be coupled to beads via
free thiol groups of the beads and used to quantify the number of
free thiols present in a bead and/or track a bead.
In some cases, addition of moieties to a gel bead after gel bead
formation may be advantageous. For example, addition of a species
after gel bead formation may avoid loss of the species during chain
transfer termination that can occur during polymerization.
Moreover, smaller precursors (e.g., monomers or cross linkers that
do not comprise side chain groups and linked moieties) may be used
for polymerization and can be minimally hindered from growing chain
ends due to viscous effects. In some cases, functionalization after
gel bead synthesis can minimize exposure of species (e.g.,
oligonucleotides) to be loaded with potentially damaging agents
(e.g., free radicals) and/or chemical environments. In some cases,
the generated gel may possess an upper critical solution
temperature (UCST) that can permit temperature driven swelling and
collapse of a bead. Such functionality may aid in species (e.g., a
primer, a P5 primer) infiltration into the bead during subsequent
functionalization of the bead with the species. Post-production
functionalization may also be useful in controlling loading ratios
of species in beads, such that, for example, the variability in
loading ratio is minimized. Also, species loading may be performed
in a batch process such that a plurality of beads can be
functionalized with the species in a single batch.
In some cases, acrydite moieties linked to precursors, another
species linked to a precursor, or a precursor itself comprise a
labile bond, such as, for example, chemically, thermally, or
photo-sensitive bonds e.g., disulfide bonds, UV sensitive bonds, or
the like. Once acrydite moieties or other moieties comprising a
labile bond are incorporated into a bead, the bead may also
comprise the labile bond. The labile bond may be, for example,
useful in reversibly linking (e.g., covalently linking) species
(e.g., barcodes, primers, etc.) to a bead. In some cases, a
thermally labile bond may include a nucleic acid hybridization
based attachment, e.g., where an oligonucleotide is hybridized to a
complementary sequence that is attached to the bead, such that
thermal melting of the hybrid releases the oligonucleotide, e.g., a
barcode containing sequence, from the bead or microcapsule.
Moreover, the addition of multiple types of labile bonds to a gel
bead may result in the generation of a bead capable of responding
to varied stimuli. Each type of labile bond may be sensitive to an
associated stimulus (e.g., chemical stimulus, light, temperature,
etc.) such that release of species attached to a bead via each
labile bond may be controlled by the application of the appropriate
stimulus. Such functionality may be useful in controlled release of
species from a gel bead. In some cases, another species comprising
a labile bond may be linked to a gel bead after gel bead formation
via, for example, an activated functional group of the gel bead as
described above. As will be appreciated, barcodes that are
releasably, cleavably or reversibly attached to the beads described
herein include barcodes that are released or releasable through
cleavage of a linkage between the barcode molecule and the bead, or
that are released through degradation of the underlying bead
itself, allowing the barcodes to be accessed or accessible by other
reagents, or both. In general, the barcodes that are releasable as
described herein, may generally be referred to as being
activatable, in that they are available for reaction once released.
Thus, for example, an activatable barcode may be activated by
releasing the barcode from a bead (or other suitable type of
partition described herein). As will be appreciated, other
activatable configurations are also envisioned in the context of
the described methods and systems. In particular, reagents may be
provided releasably attached to beads, or otherwise disposed in
partitions, with associated activatable groups, such that once
delivered to the desired set of reagents, e.g., through
co-partitioning, the activatable group may be reacted with the
desired reagents. Such activatable groups include caging groups,
removable blocking or protecting groups, e.g., photolabile groups,
heat labile groups, or chemically removable groups.
In addition to thermally cleavable bonds, disulfide bonds and UV
sensitive bonds, other non-limiting examples of labile bonds that
may be coupled to a precursor or bead include an ester linkage
(e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal
diol linkage (e.g., cleavable via sodium periodate), a Diels-Alder
linkage (e.g., cleavable via heat), a sulfone linkage (e.g.,
cleavable via a base), a silyl ether linkage (e.g., cleavable via
an acid), a glycosidic linkage (e.g., cleavable via an amylase), a
peptide linkage (e.g., cleavable via a protease), or a
phosphodiester linkage (e.g., cleavable via a nuclease (e.g.,
DNAase)).
A bead may be linked to a varied number of acrydite moieties. For
example, a bead may comprise about 1, 10, 100, 1000, 10000, 100000,
1000000, 10000000, 100000000, 1000000000, or 10000000000 acrydite
moieties linked to the beads. In other examples, a bead may
comprise at least 1, 10, 100, 1000, 10000, 100000, 1000000,
10000000, 100000000, 1000000000, or 10000000000 acrydite moieties
linked to the beads. For example, a bead may comprise about 1, 10,
100, 1000, 10000, 100000, 1000000, 10000000, 100000000, 1000000000,
or 10000000000 oligonucleotides covalently linked to the beads,
such as via an acrydite moiety. In other examples, a bead may
comprise at least 1, 10, 100, 1000, 10000, 100000, 1000000,
10000000, 100000000, 1000000000, or 10000000000 oligonucleotides
covalently linked to the beads, such as via an acrydite moiety.
Species that do not participate in polymerization may also be
encapsulated in beads during bead generation (e.g., during
polymerization of precursors). Such species may be entered into
polymerization reaction mixtures such that generated beads comprise
the species upon bead formation. In some cases, such species may be
added to the gel beads after formation. Such species may include,
for example, oligonucleotides, species necessary for a nucleic acid
amplification reaction (e.g., primers, polymerases, dNTPs,
co-factors (e.g., ionic co-factors)) including those described
herein, species necessary for enzymatic reactions (e.g., enzymes,
co-factors, substrates), or species necessary for a nucleic acid
modification reaction such as polymerization, ligation, or
digestion. Trapping of such species may be controlled by the
polymer network density generated during polymerization of
precursors, control of ionic charge within the gel bead (e.g., via
ionic species linked to polymerized species), or by the release of
other species. Encapsulated species may be released from a bead
upon bead degradation and/or by application of a stimulus capable
of releasing the species from the bead.
Beads may be of uniform size or heterogeneous size. In some cases,
the diameter of a bead may be about 5 .mu.m, 10 .mu.m, 20 .mu.m, 30
.mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m,
75 .mu.m, 80 .mu.m, 90 .mu.m, 100 .mu.m, 250 .mu.m, 500 .mu.m, or 1
mm. In some cases, a bead may have a diameter of at least about 5
.mu.m, 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m,
60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 90 .mu.m, 100
.mu.m, 250 .mu.m, 500 .mu.m, 1 mm, or more. In some cases, a bead
may have a diameter of less than about 5 .mu.m, 10 .mu.m, 20 .mu.m,
30 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m, 60 .mu.m, 65 .mu.m, 70
.mu.m, 75 .mu.m, 80 .mu.m, 90 .mu.m, 100 .mu.m, 250 .mu.m, 500
.mu.m, or 1 mm. In some cases, a bead may have a diameter in the
range of about 40-75 .mu.m, 30-75 .mu.m, 20-75 .mu.m, 40-85 .mu.m,
40-95 .mu.m, 20-100 .mu.m, 10-100 .mu.m, 1-100 .mu.m, 20-250 .mu.m,
or 20-500 .mu.m.
In certain preferred aspects, the beads are provided as a
population of beads having a relatively monodisperse size
distribution. As will be appreciated, in some applications, where
it is desirable to provide relatively consistent amounts of
reagents within partitions, maintaining relatively consistent bead
characteristics, such as size, contributes to that overall
consistency. In particular, the beads described herein may have
size distributions that have a coefficient of variation in their
cross-sectional dimensions of less than 50%, less than 40%, less
than 30%, less than 20%, and in some cases less than 15%, less than
10%, or even less than 5%.
Beads may be of a regular shape or an irregular shape. Examples of
bead shapes include spherical, non-spherical, oval, oblong,
amorphous, circular, cylindrical, and homologs thereof.
Degradable Beads
In addition to, or as an alternative to the cleavable linkages
between the beads and the associated molecules, e.g., barcode
containing oligonucleotides, described above, the beads may be
degradable, disruptable, or dissolvable spontaneously or upon
exposure to one or more stimuli (e.g., temperature changes, pH
changes, exposure to particular chemical species or phase, exposure
to light, reducing agent, etc.). In some cases, a bead may be
dissolvable, such that material components of the beads are
solubilized when exposed to a particular chemical species or an
environmental changes, such as, for example, temperature, or pH.
For example, a gel bead may be degraded or dissolved at elevated
temperature and/or in basic conditions. In some cases, a bead may
be thermally degradable such that when the bead is exposed to an
appropriate change in temperature (e.g., heat), the bead degrades.
Degradation or dissolution of a bead bound to a species (e.g., a
nucleic acid species) may result in release of the species from the
bead.
A degradable bead may comprise one or more species with a labile
bond such that when the bead/species is exposed to the appropriate
stimuli, the bond is broken and the bead degrades. The labile bond
may be a chemical bond (e.g., covalent bond, ionic bond) or may be
another type of physical interaction (e.g., van der Waals
interactions, dipole-dipole interactions, etc.). In some cases, a
crosslinker used to generate a bead may comprise a labile bond.
Upon exposure to the appropriate conditions, the labile bond is
broken and the bead is degraded. For example, a polyacrylamide gel
bead may comprise cystamine crosslinkers. Upon exposure of the bead
to a reducing agent, the disulfide bonds of the cystamine are
broken and the bead is degraded.
A degradable bead may be useful in more quickly releasing an
attached species (e.g., an oligonucleotide, a barcode sequence)
from the bead when the appropriate stimulus is applied to the bead.
For example, for a species bound to an inner surface of a porous
bead or in the case of an encapsulated species, the species may
have greater mobility and accessibility to other species in
solution upon degradation of the bead. In some cases, a species may
also be attached to a degradable bead via a degradable linker
(e.g., disulfide linker). The degradable linker may respond to the
same stimuli as the degradable bead or the two degradable species
may respond to different stimuli. For example, a barcode sequence
may be attached, via a disulfide bond, to a polyacrylamide bead
comprising cystamine. Upon exposure of the barcoded-bead to a
reducing agent, the bead degrades and the barcode sequence is
released upon breakage of both the disulfide linkage between the
barcode sequence and the bead and the disulfide linkages of the
cystamine in the bead.
A degradable bead may be introduced into a partition, such as a
droplet of an emulsion or a well, such that the bead degrades
within the partition and any associated species are released within
the droplet when the appropriate stimulus is applied. The free
species may interact with other species. For example, a
polyacrylamide bead comprising cystamine and linked, via a
disulfide bond, to a barcode sequence, may be combined with a
reducing agent within a droplet of a water-in-oil emulsion. Within
the droplet, the reducing agent breaks the various disulfide bonds
resulting in bead degradation and release of the barcode sequence
into the aqueous, inner environment of the droplet. In another
example, heating of a droplet comprising a bead-bound barcode
sequence in basic solution may also result in bead degradation and
release of the attached barcode sequence into the aqueous, inner
environment of the droplet.
As will be appreciated from the above disclosure, while referred to
as degradation of a bead, in many instances as noted above, that
degradation may refer to the disassociation of a bound or entrained
species from a bead, both with and without structurally degrading
the physical bead itself. For example, entrained species may be
released from beads through osmotic pressure differences due to,
for example, changing chemical environments. By way of example,
alteration of bead pore sizes due to osmotic pressure differences
can generally occur without structural degradation of the bead
itself. In some cases, an increase in pore size due to osmotic
swelling of a bead can permit the release of entrained species
within the bead. In other cases, osmotic shrinking of a bead may
cause a bead to better retain an entrained species due to pore size
contraction.
As will be appreciated, where degradable beads are provided, it may
be desirable to avoid exposing such beads to the stimulus or
stimuli that cause such degradation prior to the desired time, in
order to avoid premature bead degradation and issues that arise
from such degradation, including for example poor flow
characteristics, clumping and aggregation. By way of example, where
beads comprise reducible cross-linking groups, such as disulfide
groups, it will be desirable to avoid contacting such beads with
reducing agents, e.g., DTT or other disulfide cleaving reagents. In
such cases, treatments to the beads described herein will, in some
cases be provided to be free of reducing agents, such as DTT.
Because reducing agents are often provided in commercial enzyme
preparations, it is often desirable to provide reducing agent free
(or DTT free) enzyme preparations in treating the beads described
herein. Examples of such enzymes include, e.g., polymerase enzyme
preparations, ligase enzyme preparations, as well as many other
enzyme preparations that may be used to treat the beads described
herein. By "reducing agent free" or "DTT free" preparations means
that the preparation will have less than 1/10th, less than
1/50.sup.th, and even less than 1/100.sup.th of the lower ranges
for such materials used in degrading the beads. For example, for
DTT, the reducing agent free preparation will typically have less
than 0.01 mM, 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less
than 0.0001 mM DTT or less. In many cases, the amount of DTT will
be undetectable.
Methods for Degrading Beads
In some cases, a stimulus may be used to trigger degrading of the
bead, which may result in the release of contents from the bead.
Generally, a stimulus may cause degradation of the bead structure,
such as degradation of the covalent bonds or other types of
physical interaction. These stimuli may be useful in inducing a
bead to degrade and/or to release its contents. Examples of stimuli
that may be used include chemical stimuli, thermal stimuli, light
stimuli and any combination thereof, as described more fully
below.
Numerous chemical triggers may be used to trigger the degradation
of beads. Examples of these chemical changes may include, but are
not limited to pH-mediated changes to the integrity of a component
within the bead, degradation of a component of a bead via cleavage
of cross-linked bonds, and depolymerization of a component of a
bead.
In some embodiments, a bead may be formed from materials that
comprise degradable chemical crosslinkers, such as BAC or
cystamine. Degradation of such degradable crosslinkers may be
accomplished through a number of mechanisms. In some examples, a
bead may be contacted with a chemical degrading agent that may
induce oxidation, reduction or other chemical changes. For example,
a chemical degrading agent may be a reducing agent, such as
dithiothreitol (DTT). Additional examples of reducing agents may
include .beta.-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane
(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP),
or combinations thereof A reducing agent may degrade the disulfide
bonds formed between gel precursors forming the bead, and thus,
degrade the bead. In other cases, a change in pH of a solution,
such as an increase in pH, may trigger degradation of a bead. In
other cases, exposure to an aqueous solution, such as water, may
trigger hydrolytic degradation, and thus degrading the bead.
Beads may also be induced to release their contents upon the
application of a thermal stimulus. A change in temperature can
cause a variety of changes to a bead. For example, heat can cause a
solid bead to liquefy. A change in heat may cause melting of a bead
such that a portion of the bead degrades. In other cases, heat may
increase the internal pressure of the bead components such that the
bead ruptures or explodes. Heat may also act upon heat-sensitive
polymers used as materials to construct beads.
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable agent to degrade beads. In some
embodiments, changes in temperature or pH may be used to degrade
thermo-sensitive or pH-sensitive bonds within beads. In some
embodiments, chemical degrading agents may be used to degrade
chemical bonds within beads by oxidation, reduction or other
chemical changes. For example, a chemical degrading agent may be a
reducing agent, such as DTT, wherein DTT may degrade the disulfide
bonds formed between a crosslinker and gel precursors, thus
degrading the bead. In some embodiments, a reducing agent may be
added to degrade the bead, which may or may not cause the bead to
release its contents. Examples of reducing agents may include
dithiothreitol (DTT), .beta.-mercaptoethanol,
(2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA),
tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. The
reducing agent may be present at 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10
mM. The reducing agent may be present at more than 0.1 mM, 0.5 mM,
1 mM, 5 mM, 10 mM, or more. The reducing agent may be present at
less than 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.
Timing of Degrading Step
Beads may be degraded to release contents attached to and contained
within the bead. This degrading step may occur simultaneously as
the sample is combined with the bead. This degrading step may occur
simultaneously when the sample is combined with the bead within a
fluidic droplet that may be formed in a microfluidic device. This
degrading step may occur after the sample is combined with the bead
within a fluidic droplet that may be formed in a microfluidic
device. As will be appreciated, in many applications, the degrading
step may not occur.
The reducing agent may be combined with the sample and then with
the bead. In some cases, the reducing agent may be introduced to a
microfluidic device as the same time as the sample. In some cases,
the reducing agent may be introduced to a microfluidic device after
the sample is introduced. In some cases, the sample may be mixed
with the reducing agent in a microfluidic device and then contacted
with the gel bead in the microfluidic device. In some embodiments,
the sample may be pre-mixed with the reducing agent and then added
to the device and contacted with the gel bead.
A degradable bead may degrade instantaneously upon application of
the appropriate stimuli. In other cases, degradation of the bead
may occur over time. For example, a bead may degrade upon
application of an appropriate stimulus instantaneously or within
about 0, 0.01, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11, 12, 13, 14,
15 or 20 minutes. In other examples, a bead may degrade upon
application of a proper stimulus instantaneously or within at most
about 0, 0.01, 0.1, 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11, 12, 13, 14,
15 or 20 minutes.
Beads may also be degraded at different times, relative to
combining with a sample. For example, the bead may be combined with
the sample and subsequently degraded at a point later in time. The
time between combining the sample with the bead and subsequently
degrading the bead may be about 0.0001, 0.001, 0.01, 1, 10, 30, 60,
300, 600, 1800, 3600, 18000, 36000, 86400, 172800, 432000, or
864000 seconds. The time between combining the sample with the bead
and subsequently degrading the bead may be more than about 0.0001,
0.001, 0.01, 1, 10, 30, 60, 300, 600, 1800, 3600, 18000, 36000,
86400, 172800, 432000, 864000 seconds or more. The time between
combining the sample with the bead and subsequently degrading the
bead may be less than about 0.0001, 0.001, 0.01, 1, 10, 30, 60,
300, 600, 1800, 3600, 18000, 36000, 86400, 172800, 432000, or
864000 seconds.
Preparing Beads Pre-Functionalized with Oligonucleotides
The beads described herein may be produced using a variety of
methods. Suitable beads are described in U.S. Patent Application
Publication No. 20140378350, filed Jun. 26, 2014, the contents of
which are incorporated herein by reference. In some cases, beads
may be formed from a liquid containing molecular precursors (e.g.
linear polymers, monomers, cross-linkers). The liquid is then
subjected to a polymerization reaction, and thereby hardens or gels
into a bead (or gel bead). The liquid may also contain entities
such as oligonucleotides that become incorporated into the bead
during polymerization. This incorporation may be via covalent or
non-covalent association with the bead. For example, in some cases,
the oligonucleotides may be entrained within a bead during
formation. Alternatively, they may be coupled to the bead or the
bead framework either during formation or following formation.
Often, the oligonucleotides are connected to an acrydite moiety
that becomes cross-linked to the bead during the polymerization
process. In some cases, the oligonucleotides are attached to the
acrydite moiety by a disulfide linkage. As a result, a composition
comprising a bead-acrydite-S--S-oligonucleotide linkage is
formed.
In one exemplary process, functionalized beads may be generated by
mixing a plurality of polymers and/or monomers with one or more
oligonucleotides, such as, for example, one or more
oligonucleotides that comprises a primer (e.g., a universal primer,
a sequencing primer). The polymers and/or monomers may comprise
acrylamide and may be crosslinked such that disulfide bonds form
between the polymers and/or monomers, resulting in the formation of
hardened beads. The oligonucleotides may be covalently linked to
the plurality of polymers and/or monomers during the formation of
the hardened beads (e.g., contemporaneously) or may be covalently
linked to the plurality of polymers and/or monomers after the
formation of the hardened beads (e.g., sequentially). In some
cases, the oligonucleotides may be linked to the beads via an
acrydite moiety.
In most cases, a population of beads is pre-functionalized with the
identical oligonucleotide such as a universal primer or primer
binding site. In some cases, the beads in a population of beads are
pre-functionalized with multiple different oligonucleotides. These
oligonucleotides may optionally include any of a variety of
different functional sequences, e.g., for use in subsequent
processing or application of the beads. Functional sequences may
include, e.g., primer sequences, such as targeted primer sequences,
universal primer sequences, e.g., primer sequences that are
sufficiently short to be able to hybridize to and prime extension
from large numbers of different locations on a sample nucleic acid,
or random primer sequences, attachment or immobilization sequences,
ligation sequences, hairpin sequences, tagging sequences, e.g.,
barcodes or sample index sequences, or any of a variety of other
nucleotide sequences.
By way of example, in some cases, the universal primer (e.g., P5 or
other suitable primer) may be used as a primer on each bead, to
attach additional content (e.g., barcodes, random N-mers, other
functional sequences) to the bead. In some cases, the universal
primer (e.g., P5) may also be compatible with a sequencing device,
and may later enable attachment of a desired strand to a flow cell
within the sequencing device. For example, such attachment or
immobilization sequences may provide a complementary sequence to
oligonucleotides that are tethered to the surface of a flow cell in
a sequencing device, to allow immobilization of the sequences to
that surface for sequencing. Alternatively, such attachments
sequences may additionally be provided within, or added to the
oligonucleotide sequences attached to the beads. In some cases, the
beads and their attached species may be provided to be compatible
with subsequent analytical process, such as sequencing devices or
systems. In some cases, more than one primer may be attached to a
bead and more than one primer may contain a universal sequence, in
order to, for example, allow for differential processing of the
oligonucleotide as well as any additional sequences that are
coupled to that sequence, in different sequential or parallel
processing steps, e.g., a first primer for amplification of a
target sequence, with a second primer for sequencing the amplified
product. For example, in some cases, the oligonucleotides attached
to the beads will comprise a first primer sequence for conducting a
first amplification or replication process, e.g., extending the
primer along a target nucleic acid sequence, in order to generate
an amplified barcoded target sequence(s). By also including a
sequencing primer within the oligonucleotides, the resulting
amplified target sequences will include such primers, and be
readily transferred to a sequencing system. For example, in some
cases, e.g., where one wishes to sequence the amplified targets
using, e.g., an Illumina sequencing system, an R1 primer or primer
binding site may also be attached to the bead.
Entities incorporated into the beads may include oligonucleotides
having any of a variety of functional sequences as described above.
For example, these oligonucleotides may include any one or more of
P5, R1, and R2 sequences, non cleavable 5' acrydite-P5, a cleavable
5' acrydite-SS-P5, R1c, sequencing primer, read primer, universal
primer, P5_U, a universal read primer, and/or binding sites for any
of these primers. In some cases, a primer may contain one or more
modified nucleotides nucleotide analogues, or nucleotide mimics.
For example, in some cases, the oligonucleotides may include
peptide nucleic acids (PNAs), locked nucleic acid (LNA)
nucleotides, or the like. In some cases, these oligonucleotides may
additionally or alternatively include nucleotides or analogues that
may be processed differently, in order to allow differential
processing at different steps of their application. For example, in
some cases one or more of the functional sequences may include a
nucleotide or analogue that is not processed by a particular
polymerase enzyme, thus being uncopied in a process step utilizing
that enzyme. For example, e.g., in some cases, one or more of the
functional sequence components of the oligonucleotides will
include, e.g., a uracil containing nucleotide, a nucleotide
containing a non-native base, a blocker oligonucleotide, a blocked
3' end, 3'ddCTP. As will be appreciated, sequences of any of these
entities may function as primers or primer binding sites depending
on the particular application.
Polymerization may occur spontaneously. In some cases,
polymerization may be initiated by an initiator and/or an
accelerator, by electromagnetic radiation, by temperature changes
(e.g., addition or removal of heat), by pH changes, by other
methods, and combinations thereof. An initiator may refer to a
species capable of initiating a polymerization reaction by
activating (e.g., via the generation of free radicals) one or more
precursors used in the polymerization reaction. An accelerator may
refer to a species capable of accelerating the rate at which a
polymerization reaction occurs. In some cases, an accelerator may
speed up the activation of an initiator (e.g., via the generation
of free radicals) used to then activate monomers (e.g., via the
generation of free radicals) and, thus, initiate a polymerization
reaction. In some cases, faster activation of an initiator can give
rise to faster polymerization rates. In some cases, though,
acceleration may also be achieved via non-chemical means such as
thermal (e.g., addition and removal of heat) means, various types
of radiative means (e.g., visible light, UV light, etc.), or any
other suitable means. To create droplets containing molecular
precursors, which may then polymerize to form hardened beads, an
emulsion technique may be employed. For example, molecular
precursors may be added to an aqueous solution. The aqueous
solution may then be emulsified with an oil (e.g., by agitation,
microfluidic droplet generator, or other method). The molecular
precursors may then be polymerized in the emulsified droplets to
form the beads.
An emulsion may be prepared, for example, by any suitable method,
including methods known in the art, such as bulk shaking, bulk
agitation, flow focusing, and microsieve (See e.g., Weizmann et
al., Nature Methods, 2006, 3(7):545-550; Weitz et al. U.S. Pub. No.
2012/0211084). In some cases, an emulsion may be prepared using a
microfluidic device. In some cases, water-in-oil emulsions may be
used. These emulsions may incorporate fluorosurfactants such as
Krytox FSH with a PEG-containing compound such as bis krytox peg
(BKP). In some cases, oil-in-water emulsions may be used. In some
cases, polydisperse emulsions may be formed. In some cases,
monodisperse emulsions may be formed. In some cases, monodisperse
emulsions may be formed in a microfluidic flow focusing device.
(Gartecki et al., Applied Physics Letters, 2004,
85(13):2649-2651).
In at least one example, a microfluidic device for making the beads
may contain channel segments that intersect at a single cross
intersection that combines two or more streams of immiscible
fluids, such as an aqueous solution containing molecular precursors
and an oil.
Combining two immiscible fluids at a single cross intersection may
cause fluidic droplets to form. The size of the fluidic droplets
formed may depend upon the flow rate of the fluid streams entering
the fluidic cross, the properties of the two fluids, and the size
of the microfluidic channels. Initiating polymerization after
formation of fluidic droplets exiting the fluidic cross may cause
hardened beads to form from the fluidic droplets. Examples of
microfluidic devices, channel networks and systems for generating
droplets, both for bead formation and for partitioning beads into
discrete droplets as discussed elsewhere herein, are described for
example in U.S. Pub. No. 20150292988, and incorporated herein by
reference in its entirety for all purposes.
To manipulate when individual molecular precursors, oligomers, or
polymers begin to polymerize to form a hardened bead, an initiator
and/or accelerator may be added at different points in the bead
formation process. An accelerator may be an agent which may
initiate the polymerization process (e.g., in some cases, via
activation of a polymerization initiator) and thus may reduce the
time for a bead to harden. In some cases, a single accelerator or a
plurality of accelerators may be used for polymerization. Careful
tuning of acceleration can be important in achieving suitable
polymerization reactions. For example, if acceleration is too fast,
weight and excessive chain transfer events may cause poor gel
structure and low loading of any desired species. If acceleration
is too slow, high molecular weight polymers can generate trapped
activation sites (e.g., free radicals) due to polymer entanglement
and high viscosities. High viscosities can impede diffusion of
species intended for bead loading, resulting in low to no loading
of the species. Tuning of accelerator action can be achieved, for
example, by selecting an appropriate accelerator, an appropriate
combination of accelerators, or by selecting the appropriate
accelerator(s) and any stimulus (e.g., heat, electromagnetic
radiation (e.g., light, UV light), another chemical species, etc.)
capable of modulating accelerator action. Tuning of initiator
action may also be achieved in analogous fashion.
An accelerator may be water-soluble, oil-soluble, or may be both
water-soluble and oil-soluble. For example, an accelerator may be
tetramethylethylenediamine (TMEDA or TEMED),
dimethylethylenediamine, N,N, N,'N'-tetramethylmethanediamine,
N,N'-dimorpholinomethane, or
N,N,N',N'-Tetrakis(2-Hydroxypropyl)ethylenediamine Azo-based
initiators may be used in the absence of TEMED and APS and can
function as thermal based initiators. A thermal based initiator can
activate species (e.g., via the generation of free radicals)
thermally and, thus, the rate of initiator action can be tuned by
temperature and/or the concentration of the initiator. A
polymerization accelerator or initiator may include functional
groups including phosphonate, sulfonate, carboxylate, hydroxyl,
albumin binding moieties, N-vinyl groups, and phospholipids. A
polymerization accelerator or initiator may be a low molecular
weight monomeric-compound. An accelerator or initiator may be a)
added to the oil prior to droplet generation, b) added in the line
after droplet generation, c) added to the outlet reservoir after
droplet generation, or d) combinations thereof.
Polymerization may also be initiated by electromagnetic radiation.
Certain types of monomers, oligomers, or polymers may contain
light-sensitive properties. Thus, polymerization may be initiated
by exposing such monomers, oligomers, or polymers to UV light,
visible light, UV light combined with a sensitizer, visible light
combined with a sensitizer, or combinations thereof. An example of
a sensitizer may be riboflavin.
The time for a bead to completely polymerize or harden may vary
depending on the size of the bead, whether an accelerator may be
added, when an accelerator may be added, the type of initiator,
when electromagnetic radiation may be applied, the temperature of
solution, the polymer composition, the polymer concentration, and
other relevant parameters. For example, polymerization may be
complete after about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 minutes. Polymerization may be complete after more
than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 minutes or more. Polymerization may be complete in less than
about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
minutes.
Beads may be recovered from emulsions (e.g. gel-water-oil) by
continuous phase exchange. Excess aqueous fluid may be added to the
emulsion (e.g. gel-water-oil) and the hardened beads may be
subjected to sedimentation, wherein the beads may be aggregated and
the supernatant containing excess oil may be removed. This process
of adding excess aqueous fluid followed by sedimentation and
removal of excess oil may be repeated until beads are suspended in
a given purity of aqueous buffer, with respect to the continuous
phase oil. The purity of aqueous buffer may be about 80%, 90%, 95%,
96%, 97%, 98%, or 99% (v/v). The purity of aqueous buffer may be
more than about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more (v/v).
The purity of aqueous buffer may be less than about 80%, 90%, 95%,
96%, 97%, 98%, or 99% (v/v). The sedimentation step may be repeated
about 2, 3, 4, or 5 times. The sedimentation step may be repeated
more than about 2, 3, 4, 5 times or more. The sedimentation step
may be repeated less than about 2, 3, 4, or 5 times. In some cases,
sedimentation and removal of the supernatant may also remove
un-reacted starting materials.
Examples of droplet generators may include single flow focuser,
parallel flow focuser, and microsieve membrane, such as those used
by Nanomi B. V., and others. Preferably, a microfluidic device is
used to generate the droplets.
Barcode and Random N-Mers (Introduction)
Certain applications, for example polynucleotide library
sequencing, may rely on unique identifiers ("barcodes") to identify
a sequence and, for example, to assemble a larger sequence from
sequenced fragments. Therefore, it may be desirable to add barcodes
to polynucleotide fragments before sequencing. In the case of
nucleic acid applications, such barcodes are typically comprised of
a relatively short sequence of nucleotides attached to a sample
sequence, where the barcode sequence is either known, or
identifiable by its location or sequence elements. In some cases, a
unique identifier may be useful for sample indexing. In some cases,
though, barcodes may also be useful in other contexts. For example,
a barcode may serve to track samples throughout processing (e.g.,
location of sample in a lab, location of sample in plurality of
reaction vessels, etc.); provide manufacturing information; track
barcode performance over time (e.g., from barcode manufacturing to
use) and in the field; track barcode lot performance over time in
the field; provide product information during sequencing and
perhaps trigger automated protocols (e.g., automated protocols
initiated and executed with the aid of a computer) when a barcode
associated with the product is read during sequencing; track and
troubleshoot problematic barcode sequences or product lots; serve
as a molecular trigger in a reaction involving the barcode, and
combinations thereof. In particularly preferred aspects, and as
alluded to above, barcode sequence segments as described herein,
can be used to provide linkage information as between two discrete
determined nucleic acid sequences. This linkage information may
include, for example, linkage to a common sample, a common reaction
vessel, e.g., a well or partition, or even a common starting
nucleic acid molecule. In particular, by attaching common barcodes
to a specific sample component, or subset of sample components
within a given reaction volume, one can attribute the resulting
sequences bearing that barcode to that reaction volume. In turn,
where the sample is allocated to that reaction volume based upon
its sample of origin, the processing steps to which it is
subsequently exposed, or on an individual molecule basis, one can
better identify the resulting sequences as having originated from
that reaction volume.
Barcodes may be generated from a variety of different formats,
including bulk synthesized polynucleotide barcodes, randomly
synthesized barcode sequences, microarray based barcode synthesis,
native nucleotides, partial complement with N-mer, random N-mer,
pseudo random N-mer, or combinations thereof. Synthesis of barcodes
is described herein, as well as in, for example, in U.S. Pub. No.
20140228255, the full disclosure of which is hereby incorporated
herein by reference in its entirety for all purposes.
As described above, oligonucleotides incorporating barcode sequence
segments, which function as a unique identifier, may also include
additional sequence segments. Such additional sequence segments may
include functional sequences, such as primer sequences, primer
annealing site sequences, immobilization sequences, or other
recognition or binding sequences useful for subsequent processing,
e.g., a sequencing primer or primer binding site for use in
sequencing of samples to which the barcode containing
oligonucleotide is attached. Further, as used herein, the reference
to specific functional sequences as being included within the
barcode containing sequences also envisioned the inclusion of the
complements to any such sequences, such that upon complementary
replication will yield the specific described sequence.
In some examples, barcodes or partial barcodes may be generated
from oligonucleotides obtained from or suitable for use in an
oligonucleotide array, such as a microarray or bead array. In such
cases, oligonucleotides of a microarray may be cleaved, (e.g.,
using cleavable linkages or moieties that anchor the
oligonucleotides to the array (such as photoclevable, chemically
cleavable, or otherwise cleavable linkages)) such that the free
oligonucleotides are capable of serving as barcodes or partial
barcodes. In some cases, barcodes or partial barcodes are obtained
from arrays are of known sequence. The use of known sequences,
including those obtained from an array, for example, may be
beneficial in avoiding sequencing errors associated with barcodes
of unknown sequence. A microarray may provide at least about
10,000,000, at least about 1,000,000, at least about 900,000, at
least about 800,000, at least about 700,000, at least about
600,000, at least about 500,000, at least about 400,000, at least
about 300,000, at least about 200,000, at least about 100,000, at
least about 50,000, at least about 10,000, at least about 1,000, at
least about 100, or at least about 10 different sequences that may
be used as barcodes or partial barcodes.
The beads provided herein may be attached to oligonucleotide
sequences that may behave as unique identifiers (e.g., barcodes).
Often, a population of beads provided herein contains a diverse
library of barcodes, wherein each bead is attached to multiple
copies of a single barcode sequence. In some cases, the barcode
sequences are pre-synthesized and/or designed with known sequences.
In some cases, each bead within the library is attached to a unique
barcode sequence. In some cases, a plurality of beads will have the
same barcode sequence attached to them. For example, in some cases
about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 50%, 75%, 80%, 90%,
95%, or 100% of the beads in a library are attached to a barcode
sequence that is identical to a barcode sequence attached to a
different bead in the library. Sometimes, about 1%, 2%, 3%, 4%, 5%,
10%, 20%, 25%, or 30% of the beads are attached to the same barcode
sequence.
The length of a barcode sequence may be any suitable length,
depending on the application. In some cases, a barcode sequence may
be about 2 to about 500 nucleotides in length, about 2 to about 100
nucleotides in length, about 2 to about 50 nucleotides in length,
about 2 to about 20 nucleotides in length, about 6 to about 20
nucleotides in length, or about 4 to 16 nucleotides in length. In
some cases, a barcode sequence is about 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 85, 90, 95, 100, 150, 200, 250, 300, 400, or 500
nucleotides in length. In some cases, a barcode sequence is greater
than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95,
100, 150, 200, 250, 300, 400, 500, 750, 1000, 5000, or 10000
nucleotides in length. In some cases, a barcode sequence is less
than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95,
100, 150, 200, 250, 300, 400, 500, 750 or 1000 nucleotides in
length.
The barcodes may be loaded into beads so that one or more barcodes
are introduced into a particular bead. In some cases, each bead may
contain the same set of barcodes. In other cases, each bead may
contain different sets of barcodes. In other cases, each bead may
comprise a set of identical barcodes. In other cases, each bead may
comprise a set of different barcodes.
The beads provided herein may be attached to oligonucleotide
sequences that are random, pseudo-random, or targeted N-mers
capable of priming a sample (e.g., genomic sample) in a downstream
process. In some cases, the same n-mer sequences will be present on
the oligonucleotides attached to a single bead or bead population.
This may be the case for targeted priming methods, e.g., where
primers are selected to target certain sequence segments within a
larger target sequence. In other cases, each bead within a
population of beads herein is attached to a large and diverse
number of N-mer sequences to, among other things, diversify the
sampling of these primers against template molecules, as such
random n-mer sequences will randomly prime against different
portions of the sample nucleic acids.
The length of an N-mer may vary. In some cases, an N-mer (e.g., a
random N-mer, a pseudo-random N-mer, or a targeted N-mer) may be
between about 2 and about 100 nucleotides in length, between about
2 and about 50 nucleotides in length, between about 2 and about 20
nucleotides in length, between about 5 and about 25 nucleotides in
length, or between about 5 and about 15 nucleotides in length. In
some cases, an N-mer (e.g., a random N-mer, a pseudo-random N-mer,
or a targeted N-mer) may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 85, 90, 95, 100, 150, 200, 250, 300, 400, or 500
nucleotides in length. In some cases, an N-mer (e.g., a random
N-mer, a pseudo-random N-mer, or targeted a N-mer) may be greater
than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95,
100, 150, 200, 250, 300, 400, 500, 750, 1000, 5000, or 10000
nucleotides in length. In some cases, an N-mer (e.g., a random
N-mer, a pseudo-random N-mer, or a targeted N-mer) may be less than
about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 85, 90, 95, 100,
150, 200, 250, 300, 400, 500, 750, or 1000 nucleotides in
length.
N-mers (including random N-mers) can be engineered for priming a
specific sample type. For example, N-mers of different lengths may
be generated for different types of sample nucleic acids or
different regions of a sample nucleic acid, such that each N-mer
length corresponds to each different type of sample nucleic acid or
each different region of a sample nucleic acid. For example, an
N-mer of one length may be generated for sample nucleic acid
originating from the genome of one species (e.g., for example, a
human genome) and an N-mer of another length may be generated for a
sample nucleic acid originating from another species (e.g., for
example, a yeast genome). In another example, an N-mer of one
length may be generated for sample nucleic acid comprising a
particular sequence region of a genome and an N-mer of another
length may be generated for a sample nucleic acid comprising
another sequence region of the genome. Moreover, in addition or as
an alternative to N-mer length, the base composition of the N-mer
(e.g., GC content of the N-mer) may also be engineered to
correspond to a particular type or region of a sample nucleic acid.
Base content may vary in a particular type of sample nucleic acid
or in a particular region of a sample nucleic acid, for example,
and, thus, N-mers of different base content may be useful for
priming different sample types of nucleic acid or different regions
of a sample nucleic acid.
Populations of beads described elsewhere herein can be generated
with an N-mer engineered for a particular sample type or particular
sample sequence region. In some cases, a mixed population of beads
(e.g., a mixture of beads comprising an N-mer engineered for one
sample type or sequence region and beads comprising another N-mer
engineered for another sample type or sequence region) with respect
to N-mer length and content may be generated. In some cases, a
population of beads may be generated, where one or more of the
beads can comprise a mixed population of N-mers engineered for a
plurality of sample types or sequence regions.
As noted previously, in some cases, the N-mers, whether random or
targeted, may comprise nucleotide analogues, mimics, or non-native
nucleotides, in order to provide primers that have improved
performance in subsequent processing steps. For example, in some
cases, it may be desirable to provide N-mer primers that have
different melting/annealing profiles when subjected to thermal
cycling, e.g., during amplification, in order to enhance the
relative priming efficiency of the n-mer sequence. In some cases,
nucleotide analogues or non-native nucleotides may be incorporated
into the N-mer primer sequences in order to alter the melting
temperature profile of the primer sequence as compared to a
corresponding primer that includes native nucleotides. In certain
cases, the primer sequences, such as the N-mer sequences described
herein, may include modified nucleotides or nucleotide analogues,
e.g., LNA bases, at one or more positions within the sequence, in
order to provide elevated temperature stability for the primers
when hybridized to a template sequence, as well as provide
generally enhanced duplex stability. In some cases, LNA nucleotides
are used in place of the A or T bases in primer synthesis to
replace those weaker binding bases with tighter binding LNA
analogues. By providing enhanced hybridizing primer sequences, one
may generate higher efficiency amplification processes using such
primers, as well as be able to operate within different temperature
regimes.
Other modifications may also be provided to the oligonucleotides
described above. For example, in some cases, the oligonucleotides
may be provided with protected termini or other regions, in order
to prevent or reduce any degradation of the oligonucleotides, e.g.,
through any present exonuclease activity. In one example, the
oligonucleotides may be provided with one or more phosphorothioate
nucleotide analogue at one or more positions within the
oligonucleotide sequence, e.g., adjacent or proximal to the 3'
and/or 5' terminal position. These phosphorothioate nucleotides
typically provide a sulfur group in place of the non-linking oxygen
in an internucleotide linkage within the oligonucleotide to reduce
or eliminate nuclease activity on the oligonucleotides, including,
e.g., 3'-5' and/or 5'-3' exonucleases. In general, phosphorothioate
analogues are useful in imparting exo and/or endonuclease
resistance to oligonucleotides that include them, including
providing protection against, e.g., 3'-5' and/or 5'-3' exonuclease
digestion of the oligonucleotides. Accordingly, in some aspects,
these one or more phosphorothioate linkages will be in one or more
of the last 5 to 10 internucleotide linkages at either the 3' or
the 5' terminus of the oligonucleotides, and preferably include one
or more of the last 3' or 5' terminal internucleotide linkage and
second to last 5' terminal internucleotide linkage, in order to
provide protection against 3'-5' or 5'-3' exonuclease activity.
Other positions within the oligonucleotides may also be provided
with phosphorothiate linkages as well. In addition to providing
such protection on the oligonucleotides that comprise the barcode
sequences (and any associated functional sequences), the above
described modifications are also useful in the context of the
blocker sequences described herein, e.g., incorporating
phosphorothioate analogues within the blocker sequences, e.g.,
adjacent or proximal to the 3' and/or 5' terminal position as well
as potentially other positions within the oligonucleotides.
Attaching Content to Pre-Functionalized Beads
A variety of content may be attached to the beads described herein,
including beads functionalized with oligonucleotides. Often,
oligonucleotides are attached, particularly oligonucleotides with
desired sequences (e.g., barcodes, random N-mers). In many of the
methods provided herein, the oligonucleotides are attached to the
beads through a primer extension reaction. Beads pre-functionalized
with primer can be contacted with oligonucleotide template.
Amplification reactions may then be performed so that the primer is
extended such that a copy of the complement of the oligonucleotide
template is attached to the primer. Other methods of attachment are
also possible such as ligation reactions.
In some cases, oligonucleotides with different sequences (or the
same sequences) are attached to the beads in separate steps. For
example, in some cases, barcodes with unique sequences are attached
to beads such that each bead has multiple copies of a first barcode
sequence on it. In a second step, the beads can be further
functionalized with a second sequence. The combination of first and
second sequences may serve as a unique barcode, or unique
identifier, attached to a bead. The process may be continued to add
additional sequences that behave as barcode sequences (in some
cases, greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 barcode
sequences are sequentially added to each bead). The beads may also
be further functionalized random N-mers that can, for example, act
as a random primer for downstream whole genome amplification
reactions.
In some cases, after functionalization with a certain
oligonucleotide sequence (e.g., barcode sequence), the beads may be
pooled and then contacted with a large population of random Nmers
that are then attached to the beads. In some cases, particularly
when the beads are pooled prior to the attachment of the random
Nmers, each bead has one barcode sequence attached to it, (often as
multiple copies), but many different random Nmer sequences attached
to it.
Limiting dilution may be used to attach oligonucleotides to beads,
such that the beads, on average, are attached to no more than one
unique oligonucleotide sequence such as a barcode. Often, the beads
in this process are already functionalized with a certain
oligonucleotide, such as primers. For example, beads functionalized
with primers (e.g., such as universal primers) and a plurality of
template oligonucleotides may be combined, often at a high ratio of
beads:template oligonucleotides, to generate a mixture of beads and
template oligonucleotides. The mixture may then be partitioned into
a plurality of partitions (e.g., aqueous droplets within a
water-in-oil emulsion), such as by a bulk emulsification process,
emulsions within plates, or by a microfluidic device, such as, for
example, a microfluidic droplet generator. In some cases, the
mixture can be partitioned into a plurality of partitions such
that, on average, each partition comprises no more than one
template oligonucleotide.
The barcodes may be loaded into the beads at an expected or
predicted ratio of barcodes per bead to be barcoded. In some cases,
the barcodes are loaded such that a ratio of about 0.0001, 0.001,
0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000,
10000, 20000, 50000, 100000, 500000, 1000000, 5000000, 10000000,
50000000, 100000000, 500000000, 1000000000, 5000000000,
10000000000, 50000000000, or 100000000000 barcodes are loaded per
bead. In some cases, the barcodes are loaded such that a ratio of
more than 0.0001, 0.001, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 200000,
300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,
2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,
9000000, 10000000, 20000000, 30000000, 40000000, 50000000,
60000000, 70000000, 80000000, 90000000, 100000000, 200000000,
300000000, 400000000, 500000000, 600000000, 700000000, 800000000,
900000000, 1000000000, 2000000000, 3000000000, 4000000000,
5000000000, 6000000000, 7000000000, 8000000000, 9000000000,
10000000000, 20000000000, 30000000000, 40000000000, 50000000000,
60000000000, 70000000000, 80000000000, 90000000000, 100000000000 or
more barcodes are loaded per bead. In some cases, the barcodes are
loaded such that a ratio of less than about 0.0001, 0.0002, 0.0003,
0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002,
0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,
50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 500000,
1000000, 5000000, 10000000, 50000000, 100000000, 500000000,
1000000000, 5000000000, 10000000000, 50000000000, or 100000000000
barcodes are loaded per bead.
Beads, including those described herein (e.g., substantially
dissolvable beads, in some cases, substantially dissolvable by a
reducing agent), may be covalently or non-covalently linked to a
plurality of oligonucleotides, wherein at least a subset of the
oligonucleotides comprises a constant region or domain (e.g., a
barcode sequence, a barcode domain, a common barcode domain, or
other sequence that is constant among the oligonucleotides of the
subset) and a variable region or domain (e.g., a random sequence, a
random N-mer, or other sequence that is variable among the
oligonucleotides of the subset). In some cases, the
oligonucleotides may be releasably coupled to a bead, as described
elsewhere herein. Oligonucleotides may be covalently or
non-covalently linked to a bead via any suitable linkage, including
types of covalent and non-covalent linkages described elsewhere
herein. In some cases, an oligonucleotide may be covalently linked
to a bead via a cleavable linkage such as, for example, a
chemically cleavable linkage (e.g., a disulfide linkage), a
photocleavable linkage, or a thermally cleavable linkage. Beads may
comprise more than about or at least about 1, 10, 50, 100, 500,
1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000,
10000000, 50000000, 100000000, 500000000, 1000000000, 5000000000,
10000000000, 50000000000, 100000000000, 500000000000, or
1000000000000 oligonucleotides comprising a constant region or
domain and a variable region or domain.
In some cases, the oligonucleotides may each comprise an identical
constant region or domain (e.g., an identical barcode sequence,
identical barcode domain, a common domain, etc.). In some cases,
the oligonucleotides may each comprise a variable domain with a
different sequence. In some cases, the percentage of the
oligonucleotides that comprise an identical constant region (or
common domain) may be at least about 0.01%, 0.1%, 1%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100%. In some cases, the percentage of the
oligonucleotides that comprise a variable region with a different
sequence may be at least about 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 100%. In some cases, the percentage of beads in a
plurality of beads that comprise oligonucleotides with different
nucleotide sequences (including those comprising a variable and
constant region or domain) is at least about 0.01%, 0.1%, 1%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100%. In some cases, the
oligonucleotides may also comprise one or more additional
sequences, such as, for example a primer binding site (e.g., a
sequencing primer binding site), a universal primer sequence (e.g.,
a primer sequence that would be expected to hybridize to and prime
one or more loci on any nucleic acid fragment of a particular
length, based upon the probability of such loci being present
within a sequence of such length) or any other desired sequence
including types of additional sequences described elsewhere
herein.
As described elsewhere herein, a plurality of beads may be
generated to form, for example, a bead library (e.g., a barcoded
bead library). In some cases, the sequence of a common domain
(e.g., a common barcode domain) or region may vary between at least
a subset of individual beads of the plurality. For example, the
sequence of a common domain or region between individual beads of a
plurality of beads may be different between 2 or more, 10 or more,
50 or more, 100 or more, 500 or more, 1000 or more, 5000 or more,
10000 or more, 50000 or more, 100000 or more, 500000 or more,
1000000 or more, 5000000 or more, 10000000 or more, 50000000 or
more, 100000000 or more, 500000000 or more, 1000000000 or more,
5000000000 or more, 10000000000 or more, 50000000000 or more, or
100000000000 or more beads of the plurality. In some cases, each
bead of a plurality of beads may comprise a different common domain
or region. In some cases, the percentage of individual beads of a
plurality of beads that comprise a different common domain or
region may be at least about 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 100%. In some cases, a plurality of beads may comprise
at least about 2, 10, 50, 100, 500, 1000, 5000, 10000, 50000,
100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000,
500000000, or more different common domains coupled to different
beads in the plurality.
As an alternative to limiting dilution (e.g., via droplets of an
emulsion), other partitioning methods may be used to attach
oligonucleotides to beads. For example, the wells of a plate may be
used. Beads comprising a primer (e.g., P5, primer linked to the
bead via acrydite and, optionally, a disulfide bond) may be
combined with a template oligonucleotide (e.g., a template
oligonucleotide comprising a barcode sequence) and amplification
reagents in the wells of a plate. Each well can comprise one or
more copies of a unique template barcode sequence and one or more
beads. Thermal cycling of the plate extends the primer, via
hybridization of the template oligonucleotide to the primer, such
that the bead comprises an oligonucleotide with a sequence
complementary to the oligonucleotide template. Thermal cycling may
continue for a desired number of cycles (e.g., at least about 1, 2,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more cycles) up until all
primers have been extended.
Upon completion of thermal cycling, the beads may be pooled into a
common vessel, washed (e.g., via centrifugation, magnetic
separation, etc.), complementary strands denatured, washed again,
and then subject to additional rounds of bulk processing if
desired. For example, a random N-mer sequence may be added to the
bead-bound oligonucleotides using the primer extension method
described above for limiting dilution.
The PCR reagents may include any suitable PCR reagents. In some
cases, dUTPs may be substituted for dTTPs during the primer
extension or other amplification reactions, such that
oligonucleotide products comprise uracil containing nucleotides
rather than thymine containing nucleotides. This uracil-containing
section of the universal sequence may later be used together with a
polymerase that will not accept or process uracil-containing
templates to mitigate undesired amplification products.
Amplification reagents may include a universal primer, universal
primer binding site, sequencing primer, sequencing primer binding
site, universal read primer, universal read binding site, or other
primers compatible with a sequencing device, e.g., an Illumina
sequencer, Ion Torrent sequencer, etc. The amplification reagents
may include P5, non cleavable 5' acrydite-P5, a cleavable 5'
acrydite-SS-P5, R1c, Biotin R1c, sequencing primer, read primer,
P5_Universal, P5_U, 52-BioR1-rc, a random N-mer sequence, a
universal read primer, etc. In some cases, a primer may contain a
modified nucleotide, a locked nucleic acid (LNA), an LNA
nucleotide, a uracil containing nucleotide, a nucleotide containing
a non-native base, a blocker oligonucleotide, a blocked 3' end,
3'ddCTP.
As described herein, in some cases oligonucleotides comprising
barcodes are partitioned such that each bead is partitioned with,
on average, less than one unique oligonucleotide sequence, less
than two unique oligonucleotide sequences, less than three unique
oligonucleotide sequences, less than four unique oligonucleotide
sequences, less than five unique oligonucleotide sequences, or less
than ten unique oligonucleotide sequences. Therefore, in some
cases, a fraction of the beads does not contain an oligonucleotide
template and therefore cannot contain an amplified oligonucleotide.
Thus, it may be desirable to separate beads comprising
oligonucleotides from beads not comprising oligonucleotides. In
some cases, this may be done using a capture moiety.
In some embodiments, a capture moiety may be used with isolation
methods such as magnetic separation to separate beads containing
barcodes from beads, which may not contain barcodes. As such, in
some cases, the amplification reagents may include capture moieties
attached to a primer or probe. Capture moieties may allow for
sorting of labeled beads from non-labeled beads to confirm
attachment of primers and downstream amplification products to a
bead. Exemplary capture moieties include biotin, streptavidin,
glutathione-S-transferase (GST), cMyc, HA, etc. The capture
moieties may be, or include, a fluorescent label or magnetic label.
The capture moiety may comprise multiple molecules of a capture
moiety, e.g., multiple molecules of biotin, streptavidin, etc. In
some cases, an amplification reaction may make use of capture
primers attached to a capture moiety (as described elsewhere
herein), such that the primer hybridizes with amplification
products and the capture moiety is integrated into additional
amplified oligonucleotides during additional cycles of the
amplification reaction. In other cases, a probe comprising a
capture moiety may be hybridized to amplified oligonucleotides
following the completion of an amplification reaction such that the
capture moiety is associated with the amplified
oligonucleotides.
A capture moiety may be a member of binding pair, such that the
capture moiety can be bound with its binding pair during
separation. For example, beads may be generated that comprise
oligonucleotides that comprise a capture moiety that is a member of
a binding pair (e.g., biotin). The beads may be mixed with capture
beads that comprise the other member of the binding pair (e.g.,
streptavidin), such that the two binding pair members bind in the
resulting mixture. The bead-capture bead complexes may then be
separated from other components of the mixture using any suitable
means, including, for example centrifugation and magnetic
separation (e.g., including cases where the capture bead is a
magnetic bead).
III. Barcode Libraries
Beads may contain one or more attached barcode sequences. The
barcode sequences attached to a single bead may be identical or
different. In some cases, each bead may be attached to about 1, 5,
10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 500000,
1000000, 5000000, 10000000, 50000000, 100000000, 500000000,
1000000000, 5000000000, 10000000000, 50000000000, or 100000000000
identical barcode sequences. In some cases, each bead may be to
about 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000,
100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000,
500000000, 1000000000, 5000000000, 10000000000, 50000000000, or
100000000000 different barcode sequences. In some cases, each bead
may be attached to at least about 1, 5, 10, 50, 100, 500, 1000,
5000, 10000, 20000, 50000, 100000, 200000, 300000, 400000, 500000,
600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000,
5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,
30000000, 40000000, 50000000, 60000000, 70000000, 80000000,
90000000, 100000000, 200000000, 300000000, 400000000, 500000000,
600000000, 700000000, 800000000, 900000000, 1000000000, 2000000000,
3000000000, 4000000000, 5000000000, 6000000000, 7000000000,
8000000000, 9000000000, 10000000000, 20000000000, 30000000000,
40000000000, 50000000000, 60000000000, 70000000000, 80000000000,
90000000000, 100000000000 or more identical barcode sequences. In
some cases, each bead may be attached to at least about 1, 5, 10,
50, 100, 500, 1000, 5000, 10000, 20000, 50000, 100000, 200000,
300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000,
2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,
9000000, 10000000, 20000000, 30000000, 40000000, 50000000,
60000000, 70000000, 80000000, 90000000, 100000000, 200000000,
300000000, 400000000, 500000000, 600000000, 700000000, 800000000,
900000000, 1000000000, 2000000000, 3000000000, 4000000000,
5000000000, 6000000000, 7000000000, 8000000000, 9000000000,
10000000000, 20000000000, 30000000000, 40000000000, 50000000000,
60000000000, 70000000000, 80000000000, 90000000000, 100000000000 or
more different barcode sequences. In some cases, each bead may be
attached to less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,
20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000,
500000, 1000000, 5000000, 10000000, 50000000, 1000000000,
5000000000, 10000000000, 50000000000, or 100000000000 identical
barcode sequences. In some cases, each bead may be attached to less
than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,
3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000,
40000, 50000, 60000, 70000, 80000, 90000, 100000, 500000, 1000000,
5000000, 10000000, 50000000, 1000000000, 5000000000, 10000000000,
50000000000, or 100000000000 different barcode sequences.
An individual barcode library may comprise one or more barcoded
beads. In some cases, an individual barcode library may comprise
about 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 20000, 50000,
100000, 500000, 1000000, 5000000, 10000000, 50000000, 100000000,
500000000, 1000000000, 5000000000, 10000000000, 50000000000, or
100000000000 individual barcoded beads. In some cases, each library
may comprise at least about 1, 5, 10, 50, 100, 500, 1000, 5000,
10000, 20000, 50000, 100000, 200000, 300000, 400000, 500000,
600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000,
5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,
30000000, 40000000, 50000000, 60000000, 70000000, 80000000,
90000000, 100000000, 200000000, 300000000, 400000000, 500000000,
600000000, 700000000, 800000000, 900000000, 1000000000, 2000000000,
3000000000, 4000000000, 5000000000, 6000000000, 7000000000,
8000000000, 9000000000, 10000000000, 20000000000, 30000000000,
40000000000, 50000000000, 60000000000, 70000000000, 80000000000,
90000000000, 100000000000 or more individual barcoded beads. In
some cases, each library may comprise less than about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,
7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000,
80000, 90000, 100000, 500000, 1000000, 5000000, 10000000, 50000000,
1000000000, 5000000000, 10000000000, 50000000000, or 100000000000
individual barcoded beads. The barcoded beads within the library
may have the same sequences or different sequences.
In some embodiments, each bead may have a unique barcode sequence.
However, the number of beads with unique barcode sequences within a
barcode library may be limited by combinatorial limits. For
example, using four different nucleotides, if a barcode is 12
nucleotides in length, than the number of unique constructs may be
limited to 4.sup.12=16777216 unique constructs. Since barcode
libraries may comprise many more beads than 1677216, there may be
some libraries with multiple copies of the same barcode. In some
embodiments, the percentage of multiple copies of the same barcode
within a given library may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 15%, 20%, 25%, 30%, 40%, or 50%. In some cases, the percentage
of multiple copies of the same barcode within a given library may
be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,
25%, 30%, 40%, 50% or more. In some cases, the percentage of
multiple copies of the same barcode within a given library may be
less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,
14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, or 50%.
In some embodiments, each bead may comprise one unique barcode
sequence but multiple different random N-mers. In some cases, each
bead may have one or more different random N-mers. Again, the
number of beads with different random N-mers within a barcode
library may be limited by combinatorial limits. For example, using
four different nucleotides, if an N-mer sequence is 12 nucleotides
in length, than the number of different constructs may be limited
to 4.sup.12=16777216 different constructs. Since barcode libraries
may comprise many more beads than 16777216, there may be some
libraries with multiple copies of the same N-mer sequence. In some
embodiments, the percentage of multiple copies of the same N-mer
sequence within a given library may be 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, or 50%. In some cases, the
percentage of multiple copies of the same N-mer sequence within a
given library may be more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the
percentage of multiple copies of the same N-mer sequence within a
given library may be less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,
40%, or 50%.
In some embodiments, the unique identifier sequence within the
barcode may be different for each primer within each bead. In some
cases, the unique identifier sequence within the barcode sequence
may be the same for each primer within each bead.
IV. Samples
Types of Samples
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable sample or species. A sample (e.g., sample
material, component of a sample material, fragment of a sample
material, etc.) or species can be, for example, any substance used
in sample processing, such as a reagent or an analyte. Exemplary
samples can include one or more of whole cells, chromosomes,
polynucleotides, organic molecules, proteins, nucleic acids,
polypeptides, carbohydrates, saccharides, sugars, lipids, enzymes,
restriction enzymes, ligases, polymerases, barcodes (e.g.,
including barcode sequences, nucleic acid barcode sequences,
barcode molecules), adaptors, small molecules, antibodies,
fluorophores, deoxynucleotide triphosphate (dNTPs),
dideoxynucleotide triphosphates (ddNTPs), buffers, acidic
solutions, basic solutions, temperature-sensitive enzymes,
pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions,
magnesium chloride, sodium chloride, manganese, aqueous buffer,
mild buffer, ionic buffer, inhibitors, oils, salts, ions,
detergents, ionic detergents, non-ionic detergents,
oligonucleotides, template nucleic acid molecules (e.g., template
oligonucleotides, template nucleic acid sequences), nucleic acid
fragments, template nucleic acid fragments (e.g., fragments of a
template nucleic acid generated from fragmenting a template nucleic
acid during fragmentation, fragments of a template nucleic acid
generated from a nucleic acid amplification reaction), nucleotides,
DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double
stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA,
cosmid DNA, chromosomal DNA, genomic DNA (gDNA), viral DNA,
bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA,
siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch
and viral RNA, proteases, locked nucleic acids in whole or part,
locked nucleic acid nucleotides, nucleases, protease inhibitors,
nuclease inhibitors, chelating agents, reducing agents, oxidizing
agents, probes, chromophores, dyes, organics, emulsifiers,
surfactants, stabilizers, polymers, water, pharmaceuticals,
radioactive molecules, preservatives, antibiotics, aptamers, and
the like. In summary, the samples that are used will vary depending
on the particular processing needs.
Samples may be derived from human and non-human sources. In some
cases, samples are derived from mammals, non-human mammals,
rodents, amphibians, reptiles, dogs, cats, cows, horses, goats,
sheep, hens, birds, mice, rabbits, insects, slugs, microbes,
bacteria, parasites, or fish. Samples may be derived from a variety
of cells, including but not limited to: eukaryotic cells,
prokaryotic cells, fungi cells, heart cells, lung cells, kidney
cells, liver cells, pancreas cells, reproductive cells, stem cells,
induced pluripotent stem cells, gastrointestinal cells, blood
cells, cancer cells, bacterial cells, bacterial cells isolated from
a human microbiome sample, etc. In some cases, a sample may
comprise the contents of a cell, such as, for example, the contents
of a single cell or the contents of multiple cells. Examples of
single cell applications of the methods and systems described
herein are set forth in U.S. Pub. No. 20140378345. Samples may also
be cell-free, such as circulating nucleic acids (e.g., DNA,
RNA).
A sample may be naturally-occurring or synthetic. A sample may be
obtained from any suitable location, including from organisms,
whole cells, cell preparations and cell-free compositions from any
organism, tissue, cell, or environment. A sample may be obtained
from environmental biopsies, aspirates, formalin fixed embedded
tissues, air, agricultural samples, soil samples, petroleum
samples, water samples, or dust samples. In some instances, a
sample may be obtained from bodily fluids, which may include blood,
urine, feces, serum, lymph, saliva, mucosal secretions,
perspiration, central nervous system fluid, vaginal fluid, or
semen. Samples may also be obtained from manufactured products,
such as cosmetics, foods, personal care products, and the like.
Samples may be the products of experimental manipulation including
recombinant cloning, polynucleotide amplification, polymerase chain
reaction (PCR) amplification, purification methods (such as
purification of genomic DNA or RNA), and synthesis reactions.
Methods of Attaching Barcodes to Samples
Barcodes (or other oligonucleotides, e.g. random N-mers) may be
attached to a sample by joining the two nucleic acid segments
together through the action of an enzyme. This may be accomplished
by primer extension, polymerase chain reaction (PCR), another type
of reaction using a polymerase, or by ligation using a ligase. See
for example, FIGS. 2A, 2B and 2C and as discussed in the
Examples.
When the ligation method is used to attach a sample to a barcode,
the samples may or may not be fragmented prior to the ligation
step. In some cases, the oligonucleotides (e.g., barcodes, random
N-mers) are attached to a sample while the oligonucleotides are
still attached to the beads. In some cases, the oligonucleotides
(e.g., barcodes, random N-mers) are attached to a sample after the
oligonucleotides are released from the beads, e.g., by cleavage of
the oligonucleotides comprising the barcodes from the beads and/or
through degradation of the beads.
The oligonucleotides may include one or more random N-mer
sequences. A collection of unique random N-mer sequences may prime
random portions of a DNA segment, thereby amplifying a sample
(e.g., a whole genome). The resulting product may be a collection
of barcoded fragments representative of the entire sample (e.g.,
genome).
The samples may or may not be fragmented before ligation to
barcoded beads. DNA fragmentation may involve separating or
disrupting DNA strands into small pieces or segments. A variety of
methods may be employed to fragment DNA including restriction
digest or various methods of generating shear forces. Restriction
digest may utilize restriction enzymes to make intentional cuts in
a DNA sequence by blunt cleavage to both strands or by uneven
cleavage to generate sticky ends. Examples of shear-force mediated
DNA strand disruption may include sonication, acoustic shearing,
needle shearing, pipetting, or nebulization. Sonication, is a type
of hydrodynamic shearing, exposing DNA sequences to short periods
of shear forces, which may result in about 700 bp fragment sizes.
Acoustic shearing applies high-frequency acoustic energy to the DNA
sample within a bowl-shaped transducer. Needle shearing generates
shear forces by passing DNA through a small diameter needle to
physically tear DNA into smaller segments. Nebulization forces may
be generated by sending DNA through a small hole of an aerosol unit
in which resulting DNA fragments are collected from the fine mist
exiting the unit.
In some cases, a ligation reaction is used to ligate
oligonucleotides to sample. One example is illustrated in FIG. 2B
(as discussed in the Examples). The ligation may involve joining
together two nucleic acid segments, such as a barcode sequence and
a sample, by catalyzing the formation of a phosphodiester bond. The
ligation reaction may include a DNA ligase, such as an E. coli DNA
ligase, a T4 DNA ligase, a mammalian ligase such as DNA ligase I,
DNA ligase III, DNA ligase IV, thermostable ligases, or the like.
The T4 DNA ligase may ligate segments containing DNA,
oligonucleotides, RNA, and RNA-DNA hybrids. The ligation reaction
may not include a DNA ligase, utilizing an alternative such as a
topoisomerase. To ligate a sample to a barcode sequence, utilizing
a high DNA ligase concentration and including PEG may achieve rapid
ligation. The optimal temperature for DNA ligase, which may be
37.degree. C., and the melting temperature of the DNA to be
ligated, which may vary, may be considered to select for a
favorable temperature for the ligation reaction. The sample and
barcoded beads may be suspended in a buffer to minimize ionic
effects that may affect ligation.
Although described in terms of ligation or direct attachment of a
barcode sequence to a sample nucleic acid component, above, the
attachment of a barcode to a sample nucleic acid, as used herein,
also encompasses the attachment of a barcode sequence to a
complement of a sample, or a copy or complement of that complement,
e.g., when the barcode is associated with a primer sequence that is
used to replicate the sample nucleic acid, as is described in
greater detail elsewhere herein. In particular, where a barcode
containing primer sequence is used in a primer extension reaction
using the sample nucleic acid (or a replicate of the sample nucleic
acid) as a template, the resulting extension product, whether a
complement of the sample nucleic acid or a duplicate of the sample
nucleic acid, will be referred to as having the barcode sequence
attached to it.
In some cases, sample is combined with the barcoded beads (either
manually or with the aid of a microfluidic device) and the combined
sample and beads are partitioned, such as in a microfluidic device.
The partitions may be aqueous droplets within a water-in-oil
emulsion. When samples are combined with barcoded beads, on average
less than two target analytes may be present in each fluidic
droplet. In some embodiments, on average, less than three target
analytes may appear per fluidic droplet. In some cases, on average,
more than two target analytes may appear per fluidic droplet. In
other cases, on average, more than three target analytes may appear
per fluidic droplet. In some cases, one or more strands of the same
target analyte may appear in the same fluidic droplet. In some
cases, less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 1000,
5000, 10000, or 100000 target analytes are present within a fluidic
droplet. In some cases, greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
50, 100, 1000, 5000, 10000, or 100000 target analytes are present
within a fluidic droplet. The partitions described herein are often
characterized by having extremely small volumes. For example, in
the case of droplet based partitions, the droplets may have overall
volumes that are less than 1000 pL, less than 900 pL, less than 800
pL, less than 700 pL, less than 600 pL, less than 500 pL, less than
400 pL, less than 300 pL, less than 200 pL, less than 100 pL, less
than 50 pL, less than 20 pL, less than 10 pL, or even less than 1
pL. Where co-partitioned with beads, it will be appreciated that
the sample fluid volume within the partitions may be less than 90%
of the above described volumes, less than 80%, less than 70%, less
than 60%, less than 50%, less than 40%, less than 30%, less than
20%, or even less than 10% the above described volumes.
When samples are combined with barcoded beads, on average less than
one bead may be present in each fluidic droplet. In some
embodiments, on average, less than two beads may be present in each
fluidic droplet. In some embodiments, on average, less than three
beads may be present per fluidic droplet. In some cases, on
average, more than one bead may be present in each fluidic droplet.
In other cases, on average, more than two beads may appear be
present in each fluidic droplet. In other cases, on average, more
than three beads may be present per fluidic droplet. In some
embodiments, a ratio of on average less than one barcoded bead per
fluidic droplet may be achieved using limiting dilution technique.
Here, barcoded beads may be diluted prior to mixing with the
sample, diluted during mixing with the sample, or diluted after
mixing with the sample.
The number of different barcodes or different sets of barcodes
(e.g., different sets of barcodes, each different set coupled to a
different bead) that are partitioned may vary depending upon, for
example, the particular barcodes to be partitioned and/or the
application. Different sets of barcodes may be, for example, sets
of identical barcodes where the identical barcodes differ between
each set. Or different sets of barcodes may be, for example, sets
of different barcodes, where each set differs in its included
barcodes. In some cases, different barcodes are partitioned by
attaching different barcodes to different beads (e.g., gel beads).
In some cases, different sets of barcodes are partitioned by
disposing each different set in a different partition. In some
cases, though a partition may comprise one or more different
barcode sets. For example, each different set of barcodes may be
coupled to a different bead (e.g., a gel bead). Each different bead
may be partitioned into a fluidic droplet, such that each different
set of barcodes is partitioned into a different fluidic droplet.
For example, about 1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000,
40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100000, 200,000,
300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,
1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000,
8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more
different barcodes or different sets of barcodes may be
partitioned. In some examples, at least about 1, 5, 10, 50, 100,
1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,
90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000,
700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000,
5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,
50000000, 100000000, or more different barcodes or different sets
of barcodes may be partitioned. In some examples, less than about
1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000,
70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000,
600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000,
4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000,
20000000, 50000000, or 100000000 different barcodes or different
sets of barcodes may be partitioned. In some examples, about 1-5,
5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000,
100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000
different barcodes or different sets of barcodes may be
partitioned.
Barcodes may be partitioned at a particular density. For example,
barcodes may be partitioned so that each partition contains about
1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000,
60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000,
500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000,
3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,
10000000, 20000000, 50000000, or 100000000 barcodes per partition.
Barcodes may be partitioned so that each partition contains at
least about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000,
50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000,
400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000,
2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000,
9000000, 10000000, 20000000, 50000000, 100000000, or more barcodes
per partition. Barcodes may be partitioned so that each partition
contains less than about 1, 5, 10, 50, 100, 1000, 10000, 20000,
30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,
300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,
1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000,
8000000, 9000000, 10000000, 20000000, 50000000, or 100000000
barcodes per partition. Barcodes may be partitioned such that each
partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000,
1000-10000, 10000-100000, 100000-1000000, 10000-1000000,
10000-10000000, or 10000-100000000 barcodes per partition. In some
cases, partitioned barcodes may be coupled to one or more beads,
such as, for example, a gel bead. In some cases, the partitions are
fluidic droplets.
Barcodes may be partitioned such that identical barcodes are
partitioned at a particular density. For example, identical
barcodes may be partitioned so that each partition contains about
1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000,
60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000,
500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000,
3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,
10000000, 20000000, 50000000, or 100000000 identical barcodes per
partition. Barcodes may be partitioned so that each partition
contains at least about 1, 5, 10, 50, 100, 1000, 10000, 20000,
30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,
300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,
1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000,
8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more
identical barcodes per partition. Barcodes may be partitioned so
that each partition contains less than about 1, 5, 10, 50, 100,
1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,
90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000,
700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000,
5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,
50000000, or 100000000 identical barcodes per partition. Barcodes
may be partitioned such that each partition contains about 1-5,
5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000,
100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000
identical barcodes per partition. In some cases, partitioned
identical barcodes may be coupled to a bead, such as, for example,
a gel bead. In some cases, the partitions are fluidic droplets.
Barcodes may be partitioned such that different barcodes are
partitioned at a particular density. For example, different
barcodes may be partitioned so that each partition contains about
1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000,
60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000,
500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000,
3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000,
10000000, 20000000, 50000000, or 100000000 different barcodes per
partition. Barcodes may be partitioned so that each partition
contains at least about 1, 5, 10, 50, 100, 1000, 10000, 20000,
30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000,
300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000,
1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000,
8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more
different barcodes per partition. Barcodes may be partitioned so
that each partition contains less than about 1, 5, 10, 50, 100,
1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,
90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000,
700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000,
5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000,
50000000, or 100000000 different barcodes per partition. Barcodes
may be partitioned such that each partition contains about 1-5,
5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000,
100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000
different barcodes per partition. In some cases, partitioned
different barcodes may be coupled to a bead, such as, for example,
a gel bead. In some cases, the partitions are fluidic droplets.
The number of partitions employed to partition barcodes or
different sets of barcodes may vary, for example, depending on the
application and/or the number of different barcodes or different
sets of barcodes to be partitioned. For example, the number of
partitions employed to partition barcodes or different sets of
barcodes may be about 5, 10, 50, 100, 250, 500, 750, 1000, 1500,
2000, 2500, 5000, 7500, or 10,000, 20000, 30000, 40000, 50000,
60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000,
500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000,
3,000,000, 4,000,000, 5,000,000, 10000000, 20000000 or more. The
number of partitions employed to partition barcodes or different
sets of barcodes may be at least about 5, 10, 50, 100, 250, 500,
750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000,
40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,
400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000,
3000000, 4000000, 5000000, 10000000, 20000000 or more. The number
of partitions employed to partition barcodes or different sets of
barcodes may be less than about 5, 10, 50, 100, 250, 500, 750,
1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000,
50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000,
500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000,
4000000, 5000000, 10000000, or 20000000. The number of partitions
employed to partition barcodes may be about 5-10000000, 55000000,
5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000,
1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000. In some
cases, the partitions may be fluidic droplets.
As described above, different barcodes or different sets of
barcodes (e.g., each set comprising a plurality of identical
barcodes or different barcodes) may be partitioned such that each
partition generally comprises a different barcode or different
barcode set. In some cases, each partition may comprise a different
set of identical barcodes, such as an identical set of barcodes
coupled to a bead (e.g., a gel bead). Where different sets of
identical barcodes are partitioned, the number of identical
barcodes per partition may vary. For example, about 100,000 or more
different sets of identical barcodes (e.g., a set of identical
barcodes attached to a bead) may be partitioned across about
100,000 or more different partitions, such that each partition
comprises a different set of identical barcodes (e.g., each
partition comprises a bead coupled to a different set of identical
barcodes). In each partition, the number of identical barcodes per
set of barcodes may be about 1,000,000 or more identical barcodes
(e.g., each partition comprises 1,000,000 or more identical
barcodes coupled to one or more beads). In some cases, the number
of different sets of barcodes may be equal to or substantially
equal to the number of partitions or may be less than the number of
partitions. Any suitable number of different barcodes or different
barcode sets, number of barcodes per partition, and number of
partitions may be combined. Thus, as will be appreciated, any of
the above-described different numbers of barcodes may be provided
with any of the above-described barcode densities per partition,
and in any of the above-described numbers of partitions.
Microfluidic Devices and Droplets
In some cases, this disclosure provides devices for making beads
and for combining beads (or other types of partitions) with
samples, e.g., for co-partitioning sample components and beads.
Such a device may be a microfluidic device (e.g., a droplet
generator). The device may be formed from any suitable material. In
some examples, a device may be formed from a material selected from
the group consisting of fused silica, soda lime glass, borosilicate
glass, poly (methyl methacrylate) PMMA, PDMS, sapphire, silicon,
germanium, cyclic olefin copolymer, polyethylene, polypropylene,
polyacrylate, polycarbonate, plastic, thermosets, hydrogels,
thermoplastics, paper, elastomers, and combinations thereof.
A device may be formed in a manner that it comprises channels for
the flow of fluids. Any suitable channels may be used. In some
cases, a device comprises one or more fluidic input channels (e.g.,
inlet channels) and one or more fluidic outlet channels. In some
embodiments, the inner diameter of a fluidic channel may be about
10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m, 60 .mu.m, 65
.mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 85 .mu.m, 90 .mu.m, 100 .mu.m,
125 .mu.m, or 150 .mu.m. In some cases, the inner diameter of a
fluidic channel may be more than 10 .mu.m, 20 .mu.m, 30 .mu.m, 40
.mu.m, 50 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m,
85 .mu.m, 90 .mu.m, 100 .mu.m, 125 .mu.m, 150 .mu.m or more. In
some embodiments, the inner diameter of a fluidic channel may be
less than about 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m,
60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 85 .mu.m, 90
.mu.m, 100 .mu.m, 125 .mu.m, or 150 .mu.m. Volumetric flow rates
within a fluidic channel may be any flow rate known in the art.
As described elsewhere herein, the microfluidic device may be
utilized to form beads by forming a fluidic droplet comprising one
or more gel precursors, one or more crosslinkers, optionally an
initiator, and optionally an aqueous surfactant. The fluidic
droplet may be surrounded by an immiscible continuous fluid, such
as an oil, which may further comprise a surfactant and/or an
accelerator.
In some embodiments, the microfluidic device may be used to combine
beads (e.g., barcoded beads or other type of first partition,
including any suitable type of partition described herein) with
sample (e.g., a sample of nucleic acids) by forming a fluidic
droplet (or other type of second partition, including any suitable
type of partition described herein) comprising both the beads and
the sample. The fluidic droplet may have an aqueous core surrounded
by an oil phase, such as, for example, aqueous droplets within a
water-in-oil emulsion. The fluidic droplet may contain one or more
barcoded beads, a sample, amplification reagents, and a reducing
agent. In some cases, the fluidic droplet may include one or more
of water, nuclease-free water, acetonitrile, beads, gel beads,
polymer precursors, polymer monomers, polyacrylamide monomers,
acrylamide monomers, degradable crosslinkers, non-degradable
crosslinkers, disulfide linkages, acrydite moieties, PCR reagents,
primers, polymerases, barcodes, polynucleotides, oligonucleotides,
nucleotides, DNA, RNA, peptide polynucleotides, complementary DNA
(cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA),
plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA,
bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA,
siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, probes, dyes,
organics, emulsifiers, surfactants, stabilizers, polymers,
aptamers, reducing agents, initiators, biotin labels, fluorophores,
buffers, acidic solutions, basic solutions, light-sensitive
enzymes, pH-sensitive enzymes, aqueous buffer, oils, salts,
detergents, ionic detergents, non-ionic detergents, and the like.
In summary, the composition of the fluidic droplet will vary
depending on the particular processing needs.
The fluidic droplets may be of uniform size or heterogeneous size.
In some cases, the diameter of a fluidic droplet may be about 1
.mu.m, 5 .mu.m, 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 45 .mu.m,
50 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 90
.mu.m, 100 .mu.m, 250 .mu.m, 500 .mu.m, or 1 mm. In some cases, a
fluidic droplet may have a diameter of at least about 1 .mu.m, 5
.mu.m, 10 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m,
60 .mu.m, 65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 90 .mu.m, 100
.mu.m, 250 .mu.m, 500 .mu.m, 1 mm or more. In some cases, a fluidic
droplet may have a diameter of less than about 1 .mu.m, 5 .mu.m, 10
.mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 45 .mu.m, 50 .mu.m, 60 .mu.m,
65 .mu.m, 70 .mu.m, 75 .mu.m, 80 .mu.m, 90 .mu.m, 100 .mu.m, 250
.mu.m, 500 .mu.m, or 1 mm. In some cases, fluidic droplet may have
a diameter in the range of about 40-75 .mu.m, 30-75 .mu.m, 20-75
.mu.m, 40-85 .mu.m, 40-95 .mu.m, 20-100 .mu.m, 10-100 .mu.m, 1-100
.mu.m, 20-250 .mu.m, or 20-500 .mu.m.
In some embodiments, the device may comprise one or more
intersections of two or more fluid input channels. For example, the
intersection may be a fluidic cross. The fluidic cross may comprise
two or more fluidic input channels and one or more fluidic outlet
channels. In some cases, the fluidic cross may comprise two fluidic
input channels and two fluidic outlet channels. In other cases, the
fluidic cross may comprise three fluidic input channels and one
fluidic outlet channel. In some cases, the fluidic cross may form a
substantially perpendicular angle between two or more of the
fluidic channels forming the cross.
In some cases, a microfluidic device may comprise a first and a
second input channel that meet at a junction that is fluidly
connected to an output channel. In some cases, the output channel
may be, for example, fluidly connected to a third input channel at
a junction. In some cases, a fourth input channel may be included
and may intersect the third input channel and outlet channel at a
junction. In some cases, a microfluidic device may comprise first,
second, and third input channels, wherein the third input channel
intersects the first input channel, the second input channel, or a
junction of the first input channel and the second input
channel.
As described elsewhere herein, the microfluidic device may be used
to generate gel beads from a liquid. For example, in some
embodiments, an aqueous fluid comprising one or more gel
precursors, one or more crosslinkers and optionally an initiator,
optionally an aqueous surfactant, and optionally an alcohol within
a fluidic input channel may enter a fluidic cross. Within a second
fluidic input channel, an oil with optionally a surfactant and an
accelerator may enter the same fluidic cross. Both aqueous and oil
components may be mixed at the fluidic cross causing aqueous
fluidic droplets to form within the continuous oil phase. Gel
precursors within fluidic droplets exiting the fluidic cross may
polymerize forming beads.
As described elsewhere herein, the microfluidic device (e.g., a
droplet generator) may be used to combine sample with beads (e.g.,
a library of barcoded beads) as well as an agent capable of
degrading the beads (e.g., reducing agent if the beads are linked
with disulfide bonds), if desired. In some embodiments, a sample
(e.g., a sample of nucleic acids) may be provided to a first
fluidic input channel that is fluidly connected to a first fluidic
cross (e.g., a first fluidic junction). Pre-formed beads (e.g.,
barcoded beads, degradable barcoded beads) may be provided to a
second fluidic input channel that is also fluidly connected to the
first fluidic cross, where the first fluidic input channel and
second fluidic input channel meet. The sample and beads may be
mixed at the first fluidic cross to form a mixture (e.g., an
aqueous mixture). In some cases, a reducing agent may be provided
to a third fluidic input channel that is also fluidly connected to
the first fluidic cross and meets the first and second fluidic
input channel at the first fluidic cross. The reducing agent can
then be mixed with the beads and sample in the first fluidic cross.
In other cases, the reducing agent may be premixed with the sample
and/or the beads before entering the microfluidic device such that
it is provided to the microfluidic device through the first fluidic
input channel with the sample and/or through the second fluidic
input channel with the beads. In other cases, no reducing agent may
be added.
In some embodiments, the sample and bead mixture may exit the first
fluidic cross through a first outlet channel that is fluidly
connected to the first fluidic cross (and, thus, any fluidic
channels forming the first fluidic cross). The mixture may be
provided to a second fluidic cross (e.g., a second fluidic
junction) that is fluidly connected to the first outlet channel. In
some cases, an oil (or other suitable immiscible) fluid may enter
the second fluidic cross from one or more separate fluidic input
channels that are fluidly connected to the second fluidic cross
(and, thus, any fluidic channels forming the cross) and that meet
the first outlet channel at the second fluidic cross. In some
cases, the oil (or other suitable immiscible fluid) may be provided
in one or two separate fluidic input channels fluidly connected to
the second fluidic cross (and, thus, the first outlet channel) that
meet the first outlet channel and each other at the second fluidic
cross. Both components, the oil and the sample and bead mixture,
may be mixed at the second fluidic cross. This mixing partitions
the sample and bead mixture into a plurality of fluidic droplets
(e.g., aqueous droplets within a water-in-oil emulsion), in which
at least a subset of the droplets that form encapsulate a barcoded
bead (e.g., a gel bead). The fluidic droplets that form may be
carried within the oil through a second fluidic outlet channel
exiting from the second fluidic cross. In some cases, fluidic
droplets exiting the second outlet channel from the second fluidic
cross may be partitioned into wells for further processing (e.g.,
thermocycling).
In many cases, it will be desirable to control the occupancy rate
of resulting droplets (or second partitions) with respect to beads
(or first partitions). Such control is described in, for example,
U.S. Pub. No. 20150292988, the full disclosure of which is
incorporated herein by reference in its entirety for all purposes.
In general, the droplets (or second partitions) will be formed such
that at least 50%, 60%, 70%, 80%, 90% or more droplets (or second
partitions) contain no more than one bead (or first partition).
Additionally, or alternatively, the droplets (or second partitions)
will be formed such that at least 50%, 60%, 70%, 80%, 90% or more
droplets (or second partitions) include exactly one bead (or first
partition). In some cases, the resulting droplets (or second
partitions) may each comprise, on average, at most about one, two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,
nineteen, or twenty beads (or first partitions). In some cases, the
resulting droplets (or second partitions) may each comprise, on
average, at least about one, two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,
sixteen, seventeen, eighteen, nineteen, twenty, or more beads (or
first partitions).
In some embodiments, samples may be pre-mixed with beads (e.g.,
degradable beads) comprising barcodes and any other reagent (e.g.,
reagents necessary for sample amplification, a reducing agent,
etc.) prior to entry of the mixture into a microfluidic device to
generate an aqueous reaction mixture. Upon entry of the aqueous
mixture to a fluidic device, the mixture may flow from a first
fluidic input channel and into a fluidic cross. In some cases, an
oil phase may enter the fluidic cross from a second fluidic input
channel (e.g., a fluidic channel perpendicular to or substantially
perpendicular to the first fluidic input channel) also fluidly
connected to the fluidic cross. The aqueous mixture and oil may be
mixed at the fluidic cross, such that an emulsion (e.g. a
gel-water-oil emulsion) forms. The emulsion can comprise a
plurality of fluidic droplets (e.g., droplets comprising the
aqueous reaction mixture) in the continuous oil phase. In some
cases, each fluidic droplet may comprise a single bead (e.g., a gel
bead attached to a set of identical barcodes), an aliquot of
sample, and an aliquot of any other reagents (e.g., reducing
agents, reagents necessary for amplification of the sample, etc.).
In some cases, though, a fluidic droplet may comprise a plurality
of beads. Upon droplet formation, the droplet may be carried via
the oil continuous phase through a fluidic outlet channel exiting
from the fluidic cross. Fluidic droplets exiting the outlet channel
may be partitioned into wells for further processing (e.g.,
thermocycling).
In cases where a reducing agent may be added to the sample prior to
entering the microfluidic device or may be added at the first
fluidic cross, the fluidic droplets formed at the second fluidic
cross may contain the reducing agent. In this case, the reducing
agent may degrade or dissolve the beads contained within the
fluidic droplet as the droplet travels through the outlet channel
leaving the second fluidic cross.
In some embodiments, a microfluidic device may contain three
discrete fluidic crosses in parallel. Fluidic droplets may be
formed at any one of the three fluidic crosses. Sample and beads
may be combined within any one of the three fluidic crosses. A
reducing agent may be added at any one of the three fluidic
crosses. An oil may be added at any one of the three fluidic
crosses.
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable oil. In some embodiments, an oil may be
used to generate an emulsion. The oil may comprise fluorinated oil,
silicon oil, mineral oil, vegetable oil, and combinations
thereof.
In some embodiments, the aqueous fluid within the microfluidic
device may also contain an alcohol. For example, an alcohol may be
glycerol, ethanol, methanol, isopropyl alcohol, pentanol, ethane,
propane, butane, pentane, hexane, and combinations thereof. The
alcohol may be present within the aqueous fluid at about 5%, 6%,
7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or
20% (v/v). In some cases, the alcohol may be present within the
aqueous fluid at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more (v/v). In some
cases, the alcohol may be present within the aqueous fluid for less
than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
17%, 18%, 19%, or 20% (v/v).
In some embodiments, the oil may also contain a surfactant to
stabilize the emulsion. For example, a surfactant may be a
fluorosurfactant, Krytox lubricant, Krytox FSH, an engineered
fluid, HFE-7500, a silicone compound, a silicon compound containing
PEG, such as bis krytox peg (BKP). The surfactant may be present at
about 0.1%, 0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,
1.8%, 1.9%, 2%, 5%, or 10% (w/w). In some cases, the surfactant may
be present at least about 0.1%, 0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,
1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 5%, 10% (w/w) or more. In some
cases, the surfactant may be present for less than about 0.1%,
0.5%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%,
5%, or 10% (w/w).
In some embodiments, an accelerator and/or initiator may be added
to the oil. For example, an accelerator may be
Tetramethylethylenediamine (TMEDA or TEMED). In some cases, an
initiator may be ammonium persulfate or calcium ions. The
accelerator may be present at about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%,
1.7%, 1.8%, 1.9%, or 2% (v/v). In some cases, the accelerator may
be present at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,
0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,
1.9%, or 2% (v/v) or more. In some cases, the accelerator may be
present for less than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,
0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,
1.8%, 1.9%, or 2% (v/v).
V. Amplification
DNA amplification is a method for creating multiple copies of small
or long segments of DNA. The methods, compositions, devices, and
kits of this disclosure may use DNA amplification to attach one or
more desired oligonucleotide sequences to individual beads, such as
a barcode sequence or random N-mer sequence. DNA amplification may
also be used to prime and extend along a sample of interest, such
as genomic DNA, utilizing a random N-mer sequence, in order to
produce a fragment of the sample sequence and couple the barcode
associated with the primer to that fragment.
For example, a nucleic acid sequence may be amplified by
co-partitioning a template nucleic acid sequence and a bead
comprising a plurality of attached oligonucleotides (e.g.,
releasably attached oligonucleotides) into a partition (e.g., a
droplet of an emulsion, a microcapsule, or any other suitable type
of partition, including a suitable type of partition described
elsewhere herein). The attached oligonucleotides can comprise a
primer sequence (e.g., a variable primer sequence such as, for
example, a random N-mer, or a targeted primer sequence such as, for
example, a targeted N-mer) that is complementary to one or more
regions of the template nucleic acid sequence and, in addition, may
also comprise a common sequence (e.g., such as a barcode sequence).
The primer sequence can be annealed to the template nucleic acid
sequence and extended (e.g., in a primer extension reaction or any
other suitable nucleic acid amplification reaction) to produce one
or more first copies of at least a portion of the template nucleic
acid, such that the one or more first copies comprises the primer
sequence and the common sequence. In cases where the
oligonucleotides comprising the primer sequence are releasably
attached to the bead, the oligonucleotides may be released from the
bead prior to annealing the primer sequence to the template nucleic
acid sequence. Moreover, in general, the primer sequence may be
extended via a polymerase enzyme (e.g., a strand displacing
polymerase enzyme as described elsewhere herein, an exonuclease
deficient polymerase enzyme as described elsewhere herein, or any
other type of suitable polymerase, including a type of polymerase
described elsewhere herein) that is also provided in the partition.
Furthermore, the oligonucleotides releasably attached to the bead
may be exonuclease resistant and, thus, may comprise one or more
phosphorothioate linkages as described elsewhere herein. In some
cases, the one or more phosphorothioate linkages may comprise a
phosphorothioate linkage at a terminal internucleotide linkage in
the oligonucleotides.
In some cases, after the generation of the one or more first
copies, the primer sequence can be annealed to one or more of the
first copies and the primer sequence again extended to produce one
or more second copies. The one or more second copies can comprise
the primer sequence, the common sequence, and may also comprise a
sequence complementary to at least a portion of an individual copy
of the one or more first copies, and/or a sequence complementary to
the variable primer sequence. The aforementioned steps may be
repeated for a desired number of cycles to produce amplified
nucleic acids.
The oligonucleotides described above may comprise a sequence
segment that is not copied during an extension reaction (such as an
extension reaction that produces the one or more first or second
copies described above). As described elsewhere herein, such a
sequence segment may comprise one or more uracil containing
nucleotides and may also result in the generation of amplicons that
form a hairpin (or partial hairpin) molecule under annealing
conditions.
In another example, a plurality of different nucleic acids can be
amplified by partitioning the different nucleic acids into separate
first partitions (e.g., droplets in an emulsion) that each comprise
a second partition (e.g., beads, including a type of bead described
elsewhere herein). The second partition may be releasably
associated with a plurality of oligonucleotides. The second
partition may comprise any suitable number of oligonucleotides
(e.g., more than 1,000 oligonucleotides, more than 10,000
oligonucleotides, more than 100,000 oligonucleotides, more than
1,000,000 oligonucleotides, more than 10,000,000 oligonucleotides,
or any other number of oligonucleotides per partition described
herein). Moreover, the second partitions may comprise any suitable
number of different barcode sequences (e.g., at least 1,000
different barcode sequences, at least 10,000 different barcode
sequences, at least 100,000 different barcode sequences, at least
1,000,000 different barcode sequences, at least 10,000,000
different barcode sequence, or any other number of different
barcode sequences described elsewhere herein).
Furthermore, the plurality of oligonucleotides associated with a
given second partition may comprise a primer sequence (e.g., a
variable primer sequence, a targeted primer sequence) and a common
sequence (e.g., a barcode sequence). Moreover, the plurality of
oligonucleotides associated with different second partitions may
comprise different barcode sequences. Oligonucleotides associated
with the plurality of second partitions may be released into the
first partitions. Following release, the primer sequences within
the first partitions can be annealed to the nucleic acids within
the first partitions and the primer sequences can then be extended
to produce one or more copies of at least a portion of the nucleic
acids with the first partitions. In general, the one or more copies
may comprise the barcode sequences released into the first
partitions.
Amplification within Droplets and Sample Indexing
Nucleic acid (e.g., DNA) amplification may be performed on contents
within fluidic droplets. As described herein, fluidic droplets may
contain oligonucleotides attached to beads. Fluidic droplets may
further comprise a sample. Fluidic droplets may also comprise
reagents suitable for amplification reactions which may include
Kapa HiFi Uracil Plus, modified nucleotides, native nucleotides,
uracil containing nucleotides, dTTPs, dUTPs, dCTPs, dGTPs, dATPs,
DNA polymerase, Taq polymerase, mutant proof reading polymerase, 9
degrees North, modified (NEB), exo (-), exo (-) Pfu, Deep Vent exo
(-), Vent exo (-), and acyclonucleotides (acyNTPS).
Oligonucleotides attached to beads within a fluidic droplet may be
used to amplify a sample nucleic acid such that the
oligonucleotides become attached to the sample nucleic acid. The
sample nucleic acids may comprise virtually any nucleic acid sought
to be analyzed, including, for example, whole genomes, exomes,
amplicons, targeted genome segments e.g., genes or gene families,
cellular nucleic acids, circulating nucleic acids, and the like,
and, as noted above, may include DNA (including gDNA, cDNA, mtDNA,
etc.) RNA (e.g., mRNA, rRNA, total RNA, etc.). Preparation of such
nucleic acids for barcoding may generally be accomplished by
methods that are readily available, e.g., enrichment or pull-down
methods, isolation methods, amplification methods etc. In order to
amplify a desired sample, such as gDNA, the random N-mer sequence
of an oligonucleotide within the fluidic droplet may be used to
prime the desired target sequence and be extended as a complement
of the target sequence. In some cases, the oligonucleotide may be
released from the bead in the droplet, as described elsewhere
herein, prior to priming For these priming and extension processes,
any suitable method of DNA amplification may be utilized, including
polymerase chain reaction (PCR), digital PCR, reverse-transcription
PCR, multiplex PCR, nested PCR, overlap-extension PCR, quantitative
PCR, multiple displacement amplification (MDA), or ligase chain
reaction (LCR). In some cases, amplification within fluidic
droplets may be performed until a certain amount of sample nucleic
acid comprising barcode may be produced. In some cases,
amplification may be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cycles. In some
cases, amplification may be performed for more than about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20
cycles, or more. In some cases, amplification may be performed for
less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 cycles.
In some cases, a sample index can be added to a sample nucleic acid
after the addition of the original barcode to the sample nucleic
acid, with or without the use of partitions or the generation of
additional partitions. In some cases, the sample index is added in
bulk. In some cases, the addition of a sample index to a sample
nucleic acid may occur prior to the addition of a barcode to the
sample nucleic acid. In some cases, the addition of a sample index
to a sample nucleic acid may occur simultaneous to or in parallel
to the addition of a sample index to the sample nucleic acid.
In alternative aspects, additional sequence segments may be ligated
to the 5' end of the partial hairpin structure where such sequence
segments are not complementary to the non-overlapped portion of the
hairpin structure. A partial hairpin structure, when subjected to
primer extension conditions, may act as its own primer and have its
5' sequence extended, as shown by the dashed arrow, until it forms
a complete or nearly complete hairpin structure, e.g., with little
or no overhang sequence. This full hairpin structure will possess
far greater duplex stability, thereby potentially negatively
impacting the ability to disrupt the hairpin structure to prime its
replication, even when employing higher affinity primers, e.g., LNA
containing primers/probes.
In some cases, a microfluidic device (e.g., a microfluidic chip)
may be useful in parallelizing sample indexing. Such a device may
comprise parallel modules each capable of adding a barcode sequence
and a sample index to nucleic acid molecules of a sample via
primers comprising both the barcode sequence and the sample index.
Each parallel module may comprise a primer set comprising a
different sample index, such that the sample processed in each
module is associated with a different sample index and set of
barcodes. For example, a microfluidic device with 8 modules may be
capable of sample indexing 8 different samples. Following barcoding
and sample indexing via attachment of the sequences to a sample
nucleic acid, bulk addition of additional sequences (e.g., R2, P7,
other barcode sequences) via, for example, serial amplification can
be used to generate sequencer-ready products as described elsewhere
herein.
A sequencer-ready product may comprise a barcode sequence that can
be used to align sequence reads and provide a sequence for a sample
nucleic acid. The sequencer-ready product may be generated, for
example, using PHASE amplification and subsequent bulk
amplification as described elsewhere herein. Moreover, the barcode
sequence may belong to a particular set of known barcode sequences.
The set of barcode sequences may be associated with a particular
sample, such that identification of the sample from which a
particular sequencing read originates can be achieved via the read
barcode sequence. Each sample can be associated with a set of known
barcode sequences, with each barcode sequence set comprising
barcode sequences that do not overlap with barcode sequence in
other barcode sets associated with other samples. Thus, the
uniqueness of a barcode sequence and its uniqueness amongst
different sets of barcode sequences may be used for
multiplexing.
In other cases, a sample index may be added to a sample nucleic
acid prior to the addition of a barcode sequence to the sample
nucleic acid. For example, a sample nucleic acid may be
pre-amplified in bulk such that resulting amplicons are attached to
a sample index sequence prior to barcoding. For example, sample may
be amplified with a primer comprising a sample index sequence such
that the sample index sequence can be attached to the sample
nucleic acid. In some cases, the primer may be a random primer
(e.g., comprising a random N-mer) and amplification may be random.
Produced amplicons that comprise the sample index can then be
barcoded using any suitable method, including barcoding methods
described herein.
Sample nucleic acid molecules can be combined into partitions
(e.g., droplets of an emulsion) with the primers described above.
In some cases, each partition can comprise a plurality of sample
nucleic acid molecules (e.g., smaller pieces of a larger nucleic
acid). In some cases, no more than one copy of a unique sample
nucleic acid molecule is present per partition. In some cases, each
partition can generally comprise primers comprising an identical
barcode sequence and a sample priming sequence (e.g., a variable
random-Nmer, a targeted N-mer), with the barcode sequence generally
differing between partitions. In such cases, each partition (and,
thus, sample nucleic acid in the partition) can be associated with
a unique barcode sequence and the unique barcode sequence can be
used to determine a sequence for the barcoded sample nucleic acid
generated in the partition.
In some cases, upon generation of barcoded sample nucleic acids,
the barcoded sample nucleic acids can be released from their
individual partitions, pooled, and subject to bulk amplification
schemes to add additional sequences (e.g., additional sequencing
primer binding sites, additional sequencer primer binding sites,
additional barcode sequences, sample index sequences) common to all
downstream sequencer-ready products. In cases where the partitions
are droplets of an emulsion, the emulsion may be broken and the
barcoded sample nucleic acids pooled. A sample index can be added
in bulk to the released, barcoded sample nucleic acids, for
example, using the serial amplification methods described herein.
Where a sample index is added in bulk, each sequencer-ready product
generated from the same sample will comprise the same sample index
that can be used to identify the sample from which the read for the
sequencer-ready product was generated. Where a sample index is
added during barcoding, each primer used for barcoding may comprise
an identical sample index sequence, such that each sequencer-ready
product generated from the same sample will comprise the same
sample index sequence.
Partitioning of sample nucleic acids to generate barcoded (or
barcoded and sample indexed) sample nucleic acids and subsequent
addition of additional sequences (e.g., including a sample index)
to the barcoded sample nucleic acids can be repeated for each
sample, using a different sample index for each sample. In some
cases, a microfluidic droplet generator may be used to partition
sample nucleic acids. In some cases, a microfluidic chip may
comprise multiple droplet generators, such that a different sample
can be processed at each droplet generator, permitting parallel
sample indexing. Via each different sample index, multiplexing
during sequencing can be achieved.
Upon the generation of sequencer-ready oligonucleotides, the
sequencer-ready oligonucleotides can then be provided to a
sequencing device for sequencing. Thus, for example, the entire
sequence provided to the sequencing device may comprise one or more
adaptors compatible with the sequencing device (e.g. P5, P7), one
or more barcode sequences, one or more primer binding sites (e.g.
Read1 (R1) sequence primer, Read2 (R2) sequencing primer, Index
primer), an N-mer sequence, a universal sequence, the sequence of
interest, and combinations thereof. The barcode sequence may be
located at either end of the sequence. In some cases, the barcode
sequence may be located between P5 and Read1 sequence primer
binding site. In other cases, the barcode sequence may be located
between P7 and Read 2 sequence primer binding site. In some cases,
a second barcode sequence may be located between P7 and Read 2
sequence primer binding site. The index sequence primer binding
site may be utilized in the sequencing device to determine the
barcode sequence.
The configuration of the various components (e.g., adaptors,
barcode sequences, sample index sequences, sample sequence, primer
binding sites, etc.) of a sequence to be provided to a sequencer
device may vary depending on, for example the particular
configuration desired and/or the order in which the various
components of the sequence is added. Any suitable configuration for
sequencing may be used and any sequences can be added to
oligonucleotides in any suitable order. Additional sequences may be
added to a sample nucleic acid prior to, during, and after
barcoding of the sample nucleic acid. For example, a P5 sequence
can be added to a sample nucleic acid during barcoding and P7 can
be added in bulk amplification following barcoding of the sample
nucleic acid. Alternatively, a P7 sequence can be added to a sample
nucleic acid during barcoding and a P5 sequence can be added in
bulk amplification following barcoding of the sample nucleic acid.
Example configurations displayed as examples herein are not
intended to be limiting. Moreover, the addition of sequence
components to an oligonucleotide via amplification is also not
meant to be limiting. Other methods, such as, for example, ligation
may also be used. Furthermore, adaptors, barcode sequences, sample
index sequences, primer binding sites, sequencer-ready products,
etc. described herein are not meant to be limiting. Any type of
oligonucleotide described herein, including sequencer-ready
products, may be generated for any suitable type of sequencing
platform (e.g., Illumina sequencing, Life Technologies Ion Torrent,
Pacific Biosciences SMRT, Roche 454 sequencing, Life Technologies
SOLiD sequencing, etc.) using methods described herein.
Sequencer-ready oligonucleotides can be generated with any adaptor
sequence suitable for a particular sequencing platform using
methods described herein. For example, sequencer-ready
oligonucleotides comprising one or more barcode sequences and P1
and A adaptor sequences useful in Life Technologies Ion Torrent
sequencing may be generated using methods described herein. In one
example, beads (e.g., gel beads) comprising an acrydite moiety
linked to a P1 sequence via a disulfide bond may be generated. A
barcode construct may be generated that comprises a P1 sequence, a
barcode sequence, and a random N-mer sequence. The barcode
construct may enter an amplification reaction (e.g., in a
partition, such as a fluidic droplet) to barcode sample nucleic
acid. Barcoded amplicons may then be subject to further
amplification in bulk to add the A sequence and any other sequence
desired, such as a sample index. Alternatively, P1 and A sequences
can be interchanged such that A is added during sample barcoding
and P1 is added in bulk. The complete sequence can then be entered
into an Ion Torrent sequencer. Other adaptor sequences (e.g., P1
adaptor sequence for Life Technologies SOLiD sequencing, A and B
adaptor sequences for Roche 454, etc.) for other sequencing
platforms can be added in analogous fashion.
Although described herein as generating partial hairpin molecules,
and in some cases, preventing formation of complete hairpins, in
some cases, it may be desirable to provide complete hairpin
fragments that include the barcode sequences described herein. In
particular, such complete hairpin molecules may be further
subjected to conventional sample preparation steps by treating the
3' and 5' end of the single hairpin molecule as one end of a double
stranded duplex molecule in a conventional sequencing workflow. In
particular, using conventional ligation steps, one could readily
attach the appropriate adapter sequences to both the 3' and 5' end
of the hairpin molecule in the same fashion as those are attached
to the 3' and 5' termini of a duplex molecule. For example, in case
of an Illumina based sequencing process, one could attach a
standard Y adapter that includes the P5 and P7 adapters and R1 and
R2 primer sequences, to one end of the hairpin as if it were one
end of a duplex molecule, using standard Illumina protocols.
VII. Digital Processor
The methods, compositions, devices, and kits of this disclosure may
be used with any suitable processor, digital processor or computer.
The digital processor may be programmed, for example, to operate
any component of a device and/or execute methods described herein.
The digital processor may be capable of transmitting or receiving
electronic signals through a computer network, such as for example,
the Internet and/or communicating with a remote computer. One or
more peripheral devices such as screen display, printer, memory,
data storage, and/or electronic display adaptors may be in
communication with the digital processor. One or more input devices
such as keyboard, mouse, or joystick may be in communication with
the digital processor. The digital processor may also communicate
with detector such that the detector performs measurements at
desired or otherwise predetermined time points or at time points
determined from feedback received from pre-processing unit or other
devices.
In one example a controller incudes a computer that serves as the
central hub for control assembly. The computer is in communication
with a display, one or more input devices (e.g., a mouse, keyboard,
camera, etc.), and optionally a printer. The control assembly, via
its computer, is in communication with one or more devices:
optionally a sample pre-processing unit, one or more sample
processing units (such as a sequence, thermocycler, or microfluidic
device), and optionally a detector. The control assembly may be
networked, for example, via an Ethernet connection. A user may
provide inputs (e.g., the parameters necessary for a desired set of
nucleic acid amplification reactions or flow rates for a
microfluidic device) into the computer, using an input device. The
inputs are interpreted by the computer, to generate instructions.
The computer communicates such instructions to the optional sample
pre-processing unit, the one or more sample processing units,
and/or the optional detector for execution.
Moreover, during operation of the optional sample pre-processing
unit, one or more sample processing units, and/or the optional
detector, each device may communicate signals back to computer.
Such signals may be interpreted and used by computer to determine
if any of the devices require further instruction. The computer may
also modulate the sample pre-processing unit such that the
components of a sample are mixed appropriately and fed, at a
desired or otherwise predetermined rate, into the sample processing
unit (such as the microfluidic device).
The computer may also communicate with a detector such that the
detector performs measurements at desired or otherwise
predetermined time points or at time points determined from
feedback received from pre-processing unit or sample processing
unit. The detector may also communicate raw data obtained during
measurements back to the computer for further analysis and
interpretation.
Analysis may be summarized in formats useful to an end user via a
display and/or printouts generated by a printer. Instructions or
programs used to control the sample pre-processing unit, the sample
processing unit, and/or the detector; data acquired by executing
any of the methods described herein; or data analyzed and/or
interpreted may be transmitted to or received from one or more
remote computers, via a network, which, for example, could be the
Internet.
In some embodiments, the method of bead formation may be executed
with the aid of a digital processor in communication with a droplet
generator. The digital processor may control the speed at which
droplets are formed or control the total number of droplets that
are generated. In some embodiments, the method of attaching samples
to barcoded beads may be executed with the aid of a digital
processor in communication with the microfluidic device.
Specifically, the digital processor may control the volumetric
amount of sample and/or beads injected into the input channels and
may also control the flow rates within the channels. In some
embodiments, the method of attaching oligonucleotides, primers, and
the like may be executed with the aid of a digital processor in
communication with a thermocycler or other programmable heating
element. Specifically, the digital processor may control the time
and temperature of cycles during ligation or amplification. In some
embodiments, the method of sequencing a sample may be executed with
the aid of a digital processor in communication with a sequencing
device.
VIII. Kits
In some cases, this disclosure provides a kit comprising a
microfluidic device, a plurality of barcoded beads, and
instructions for utilizing the microfluidic device and combining
barcoded beads with customer sample to create fluidic droplets
containing both. As specified throughout this disclosure, any
suitable sample may be incorporated into the fluidic droplets. As
described throughout this disclosure, a bead may be designed to be
degradable or non-degradable. In this case, the kit may or may not
include a reducing agent for bead degradation.
In some cases, this disclosure provides a kit comprising a
plurality of barcoded beads, suitable amplification reagents, e.g.,
optionally including one or more of polymerase enzymes, nucleoside
triphosphates or their analogues, primer sequences, buffers, and
the like, and instructions for combining barcoded beads with
customer sample. As specified throughout this disclosure, any
suitable sample may be used. As specified throughout this
disclosure, the amplification reagents may include a polymerase
that will not accept or process uracil-containing templates. A kit
of this disclosure may also provide agents to form an emulsion,
including an oil and surfactant.
IX. Applications
Barcoding Sample Materials
The methods, compositions and systems described herein are
particularly useful for attaching barcodes, and particularly
barcode nucleic acid sequences, to sample materials and components
of those sample materials. In general, this is accomplished by
partitioning sample material components into separate partitions or
reaction volumes in which are co-partitioned a plurality of
barcodes, which are then attached to sample components within the
same partition.
In an exemplary process, a first partition is provided that
includes a plurality of oligonucleotides (e.g., nucleic acid
barcode molecules) that each comprise a common nucleic acid barcode
sequence. The first partition may comprise any of a variety of
portable partitions, e.g., a bead (e.g., a degradable bead, a gel
bead), a droplet (e.g., an aqueous droplet in an emulsion), a
microcapsule, or the like, to which the oligonucleotides are
releasably attached, releasably coupled, or are releasably
associated. Moreover, any suitable number of oligonucleotides may
be included in the first partition, including numbers of
oligonucleotides per partition described elsewhere herein. For
example, the oligonucleotides may be releasably attached to,
releasably coupled to, or releasably associated with the first
partition via a cleavable linkage such as, for example, a
chemically cleavable linkage (e.g., a disulfide linkage, or any
other type of chemically cleavable linkage described herein), a
photocleavable linkage, and/or a thermally cleavable linkage. In
some cases, the first partition may be a bead and the bead may be a
degradable bead (e.g., a photodegradable bead, a chemically
degradable bead, a thermally degradable bead, or any other type of
degradable bead described elsewhere herein). Moreover, the bead may
comprise chemically-cleavable cross-linking (e.g., disulfide
cross-linking) as described elsewhere herein.
The first partition is then co-partitioned into a second partition,
with a sample material, sample material component, fragment of a
sample material, or a fragment of a sample material component. The
sample material (or component or fragment thereof) may be any
appropriate sample type, including the example sample types
described elsewhere herein. In cases where a sample material or
component of a sample material comprises one or more nucleic acid
fragments, the one or more nucleic acid fragments may be of any
suitable length, including, for example, nucleic acid fragment
lengths described elsewhere herein. The second partition may
include any of a variety of partitions, including for example,
wells, microwells, nanowells, tubes or containers, or in preferred
cases droplets (e.g., aqueous droplets in an emulsion) or
microcapsules in which the first partition may be co-partitioned.
In some cases, the first partition may be provided in a first
aqueous fluid and the sample material, sample material component,
or fragment of a sample material component may be provided in a
second aqueous fluid. During co-partitioning, the first aqueous
fluid and second aqueous fluid may be combined within a droplet
within an immiscible fluid. In some cases, the second partition may
comprise no more than one first partition. In other cases, the
second partition may comprise no more than one, two, three, four,
five, six, seven, eight, nine, or ten first partitions. In other
cases, the second partition may comprise at least one, two, three,
four, five, six, seven, eight, nine, ten, or more first
partitions.
Once co-partitioned, the oligonucleotides comprising the barcode
sequences may be released from the first partition (e.g., via
degradation of the first partition, cleaving a chemical linkage
between the oligonucleotides and the first partition, or any other
suitable type of release, including types of release described
elsewhere herein) into the second partition, and attached to the
sample components co-partitioned therewith. In some cases, the
first partition may comprise a bead and the crosslinking of the
bead may comprise a disulfide linkage. In addition, or as an
alternative, the oligonucleotides may be linked to the bead via a
disulfide linkage. In either case, the oligonucleotides may be
released from the first partition by exposing the first partition
to a reducing agent (e.g., DTT, TCEP, or any other exemplary
reducing agent described elsewhere herein).
As noted elsewhere herein, attachment of the barcodes to sample
components includes the direct attachment of the barcode
oligonucleotides to sample materials, e.g. through ligation,
hybridization, or other associations. Additionally, in many cases,
for example, in barcoding of nucleic acid sample materials (e.g.,
template nucleic acid sequences, template nucleic acid molecules),
components or fragments thereof, such attachment may additionally
comprise use of the barcode containing oligonucleotides that also
comprise as priming sequences. The priming sequence can be
complementary to at least a portion of a nucleic acid sample
material and can be extended along the nucleic acid sample
materials to create complements to such sample materials, as well
as at least partial amplification products of those sequences or
their complements.
In another exemplary process, a plurality of first partitions can
be provided that comprise a plurality of different nucleic acid
barcode sequences. Each of the first partitions can comprise a
plurality of nucleic acid barcode molecules having the same nucleic
acid barcode sequence associated therewith. Any suitable number of
nucleic acid barcode molecules may be associated with each of the
first partitions, including numbers of nucleic acid barcode
molecules per partition described elsewhere herein. The first
partitions may comprise any suitable number of different nucleic
acid barcode sequences, including, for example, at least about 2,
10, 100, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000,
5000000, 10000000, 50000000, or 1000000000, or more different
nucleic acid barcode sequences.
In some cases, the plurality of first partitions may comprise a
plurality of different first partitions where each of the different
first partitions comprises a plurality of releasably attached,
releasably coupled, or releasably associated oligonucleotides
comprising a common barcode sequence, with the oligonucleotides
associated with each different first partitions comprising a
different barcode sequence. The number of different first
partitions may be, for example, at least about 2, 10, 100, 500,
1000, 5000, 10000, 50000, 100000, 500000, 1000000, 5000000,
10000000, 50000000, or 1000000000, or more different first
partitions.
The first partitions may be co-partitioned with sample materials,
fragments of a sample material, components of a sample material, or
fragments of a component(s) of a sample material into a plurality
of second partitions. In some cases, a subset of the second
partitions may comprise the same nucleic acid barcode sequence. For
example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or more of the second partitions may comprise
the same nucleic acid barcode sequence. Moreover, the distribution
of first partitions per second partition may also vary according
to, for example, occupancy rates described elsewhere herein. In
cases where the plurality of first partitions comprises a plurality
of different first partitions, each different first partition may
be disposed within a separate second partition.
Following co-partitioning, the nucleic acid barcode molecules
associated with the first partitions can be released into the
plurality of second partitions. The released nucleic acid barcode
molecules can then be attached to the sample materials, sample
material components, fragments of a sample material, or fragments
of sample material components, within the second partitions. In the
case of barcoded nucleic acid species (e.g., barcoded sample
nucleic acid, barcoded template nucleic acid, barcoded fragments of
one or more template nucleic acid sequences, etc.), the barcoded
nucleic acid species may be sequenced as described elsewhere
herein.
In another exemplary process, an activatable nucleic acid barcode
sequence may be provided and partitioned with one or more sample
materials, components of a sample material, fragments of a sample
material, or fragments of a component(s) of a sample material into
a first partition. With the first partition, the activatable
nucleic acid barcode sequence may be activated to produce an active
nucleic acid barcode sequence. The active nucleic acid barcode
sequence can then be attached to the one or more sample materials,
components of a sample material, fragments of a sample material, or
fragments of a component(s) of a sample material.
In some cases, the activatable nucleic acid barcode sequence may be
coupled to a second partition that is also partitioned in the first
partition with the activatable nucleic acid barcode sequence. As
described elsewhere herein, an activatable nucleic acid barcode
sequence may be activated by releasing the activatable nucleic acid
barcode sequence from an associated partition (e.g., a bead). Thus,
in cases where an activatable nucleic acid barcode sequence is
associated with a second partition (e.g., a bead) that is
partitioned in a first partition (e.g., a fluidic droplet), the
activatable nucleic acid barcode sequence may be activated by
releasing the activatable nucleic acid barcode sequence from its
associated second partition. In addition, or as an alternative, an
activatable barcode may also be activated by removing a removable
blocking or protecting group from the activatable nucleic acid
barcode sequence.
In another exemplary process, a sample of nucleic acids may be
combined with a library of barcoded beads (including types of beads
described elsewhere herein) to form a mixture. In some cases, the
barcodes of the beads may, in addition to a barcode sequence, each
comprise one or more additional sequences such as, for example, a
universal sequence and/or a functional sequence (e.g., a random
N-mer or a targeted N-mer, as described elsewhere herein). The
mixture may be partitioned into a plurality of partitions, with at
least a subset of the partitions comprising at most one barcoded
bead. Within the partitions, the barcodes may be released from the
beads, using any suitable route, including types of release
described herein. A library of barcoded beads may be generated via
any suitable route, including the use of methods and compositions
described elsewhere herein. In some cases, the sample of nucleic
acids may be combined with the library of barcoded beads and/or the
resulting mixture partitioned with the aid of a microfluidic
device, as described elsewhere herein. In cases where the released
barcodes also comprise a primer sequence (e.g., such as a targeted
N-mer or a random N-mer as described elsewhere herein), the primer
sequences of the barcodes may be hybridize with the sample nucleic
acids and, if desired, an amplification reaction can be completed
in the partitions.
Polynucleotide Sequencing
Generally, the methods and compositions provided herein are useful
for preparation of oligonucleotide fragments for downstream
applications such as sequencing. In particular, these methods,
compositions and systems are useful in the preparation of
sequencing libraries. Sequencing may be performed by any available
technique. For example, sequencing may be performed by the classic
Sanger sequencing method. Sequencing methods may also include:
high-throughput sequencing, pyrosequencing, sequencing-by-ligation,
sequencing by synthesis, sequencing-by-hybridization, RNA-Seq
(Illumina), Digital Gene Expression (Helicos), next generation
sequencing, single molecule sequencing by synthesis (SMSS)
(Helicos), massively-parallel sequencing, clonal single molecule
Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing,
primer walking, and any other sequencing methods known in the
art.
For example, a plurality of target nucleic acid sequences may be
sequenced by providing a plurality of target nucleic sequences and
separating the target nucleic acid sequences into a plurality of
separate partitions. Each of the separate partitions can comprise
one or more target nucleic acid sequences and a plurality of
oligonucleotides. The separate partitions may comprise any suitable
number of different barcode sequences (e.g., at least 1,000
different barcode sequences, at least 10,000 different barcode
sequences, at least 100,000 different barcode sequences, at least
1,000,000 different barcode sequences, at least 10,000,000
different barcode sequences, or any other number of different
barcode sequences as described elsewhere herein). Moreover, the
oligonucleotides in a given partition can comprise a common barcode
sequence. The oligonucleotides and associated common barcode
sequence in a given partition can be attached to fragments of the
one or more target nucleic acids or to copies of portions of the
target nucleic acid sequences within the given partition. Following
attachment, the separate partitions can then be pooled. The
fragments of the target nucleic acids or the copies of the portions
of the target nucleic acids and attached barcode sequences can then
be sequenced.
In another example, a plurality of target nucleic acid sequences
may be sequenced by providing the target nucleic acid sequences and
separating them into a plurality of separate partitions. Each
partition of the plurality of separate partitions can include one
or more of the target nucleic acid sequences and a bead having a
plurality of attached oligonucleotides. The oligonucleotides
attached to a given bead may comprise a common barcode sequence.
The oligonucleotides associated with a bead can be attached to
fragments of the target nucleic acid sequences or to copies of
portions of the target nucleic acid sequences within a given
partition, such that the fragments or copies of the given partition
are also attached to the common barcode sequence associated with
the bead. Following attachment of the oligonucleotides to the
fragments of the target nucleic acid sequences or the copies of the
portions of the target nucleic acid sequences, the separate
partitions can then be pooled. The fragments of the target nucleic
acid sequences or the copies of the portions of the target nucleic
acid sequences and any attached barcode sequences can then be
sequenced (e.g., using any suitable sequencing method, including
those described elsewhere herein) to provide barcoded fragment
sequences or barcoded copy sequences. The barcoded fragment
sequences or barcoded copy sequences can be assembled into one or
more contiguous nucleic acid sequence based, in part, upon a
barcode portion of the barcoded fragment sequences or barcoded copy
sequences.
In some cases, varying numbers of barcoded-oligonucleotides are
sequenced. For example, in some cases about 30%-90% of the
barcoded-oligonucleotides are sequenced. In some cases, about
35%-85%, 40%-80%, 45%-75%, 55%-65%, or 50%-60% of the
barcoded-oligonucleotides s are sequenced. In some cases, at least
about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of
barcoded-oligonucleotides are sequenced. In some cases, less than
about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the
barcoded-oligonucleotides are sequenced.
In some cases, sequences from fragments are assembled to provide
sequence information for a contiguous region of the original target
polynucleotide that may be longer than the individual sequence
reads. Individual sequence reads may be about 10-50, 50-100,
100-200, 200-300, 300-400, or more nucleotides in length. Examples
of sequence assembly methods include those set forth in U.S. patent
application Ser. No. 14/752,773, filed Jun. 26, 2014.
The identities of the barcodes may serve to order the sequence
reads from individual fragments as well as to differentiate between
haplotypes. For example, when combining individual sample fragments
and barcoded beads within fluidic droplets, parental polynucleotide
fragments may be separated into different droplets. With an
increase in the number of fluidic droplets and beads within a
droplet, the likelihood of a fragment from both a maternal and
paternal haplotype contained within the same fluidic droplet
associated with the same bead may become negligibly small. Thus,
sequence reads from fragments in the same fluidic droplet and
associated with the same bead may be assembled and ordered.
In at least one example, the present disclosure provides nucleic
acid sequencing methods, systems compositions, and combinations of
these that are useful in providing myriad benefits in both sequence
assembly and read-length equivalent, but do so with very high
throughput and reduced sample preparation time and cost.
In general, the sequencing methods described herein provide for the
localized tagging or barcoding of fragments of genetic sequences.
By tagging fragments that derive from the same location within a
larger genetic sequence, one can utilize the presence of the tag or
barcode to inform the assembly process as alluded to above. In
addition, the methods described herein can be used to generate and
barcode shorter fragments from a single, long nucleic acid
molecule. Sequencing and assembly of these shorter fragments
provides a long read equivalent sequence, but without the need for
low throughput longer read-length sequencing technologies.
In accordance with the foregoing, a large genetic component, such
as a long nucleic acid fragment, e.g., 1, 10, 20, 40, 50, 75, 100,
1000 or more kb in length, a chromosomal fragment or whole
chromosome, or part of or an entire genome (e.g., genomic DNA) is
fragmented into smaller first fragments. Typically, these fragments
may be anywhere from about 1000 to about 100000 bases in length. In
certain preferred aspects, the fragments will be between about 1 kb
and about 100 kb, or between about 5 kb and about 50 kb, or from
about 10 kb to about 30 kb, and in some cases, between about 15 kb
and about 25 kb. Fragmentation of these larger genetic components
may be carried out by any of a variety of convenient available
processes, including commercially available shear based fragmenting
systems, e.g., Covaris fragmentation systems, size targeted
fragmentation systems, e.g., Blue Pippin (Sage Sciences), enzymatic
fragmentation processes, e.g., using restriction endonucleases, or
the like. As noted above, the first fragments of the larger genetic
component may comprise overlapping or non-overlapping first
fragments. Although described here as being fragmented prior to
partitioning, it will be appreciated that fragmentation may
optionally and/or additionally be performed later in the process,
e.g., following one or more amplification steps, to yield fragments
of a desired size for sequencing applications.
In preferred aspects, the first fragments are generated from
multiple copies of the larger genetic component or portions
thereof, so that overlapping first fragments are produced. In
preferred aspects, the overlapping fragments will constitute
greater than 1.times. coverage, greater than 2.times. coverage,
greater than 5.times. coverage, greater than 10.times. coverage,
greater than 20.times. coverage, greater than 40.times. coverage,
or even greater coverage of the underlying larger genetic component
or portion thereof. The first fragments are then segregated to
different reaction volumes. In some cases, the first fragments may
be separated so that reaction volumes contain one or fewer first
fragments. This is typically accomplished by providing the
fragments in a limiting dilution in solution, such that allocation
of the solution to different reaction volumes results in a very low
probability of more than one fragment being deposited into a given
reaction volume. However, in most cases, a given reaction volume
may include multiple different first fragments, and can even have
2, 5, 10, 100, 100 or even up to 10,000 or more different first
fragments in a given reaction volume. Again, achieving a desired
range of fragment numbers within individual reaction volumes is
typically accomplished through the appropriate dilution of the
solution from which the first fragments originate, based upon an
understanding of the concentration of nucleic acids in that
starting material.
The reaction volumes may include any of variety of different types
of vessels or partitions. For example, the reaction volumes may
include conventional reaction vessels, such as test tubes, reaction
wells, microwells, nanowells, or they may include less conventional
reaction volumes, such as droplets within a stabilized emulsion,
e.g., a water in oil emulsion system. In preferred aspects,
droplets are preferred as the reaction volumes for their extremely
high multiplex capability, e.g., allowing the use of hundreds of
thousands, millions, tens of millions or even more discrete
droplet/reaction volumes within a single container. Within each
reaction volume, the fragments that are contained therein are then
subjected to processing that both derives sets of overlapping
second fragments of each of the first fragments, and also provides
these second fragments with attached barcode sequences. As will be
appreciated, in preferred aspects, the first fragments are
partitioned into droplets that also contain one or more
microcapsules or beads that include the members of the barcode
library used to generate and barcode the second fragments.
In preferred aspects, the generation of these second fragments is
carried out through the introduction of primer sequences that
include the barcode sequences and that are capable of hybridizing
to portions of the first fragment and be extended along the first
fragment to provide a second fragment including the barcode
sequence. These primers may comprise targeted primer sequences,
e.g., to derive fragments that overlap specific portions of the
first fragment, or they may comprise universal priming sequences,
e.g., random primers, that will prime multiple different regions of
the first fragments to create large and diverse sets of second
fragments that span the first fragment and provide multifold
overlapping coverage. These extended primer sequences may be used
as the second fragments, or they may be further replicated or
amplified. For example, iterative priming against the extended
sequences, e.g., using the same primer containing barcoded
oligonucleotides. In certain preferred aspects, the generation of
the second sets of fragments generates the partial hairpin
replicates of portions of the first fragment, as described
elsewhere herein that each include barcode sequences, e.g., for
PHASE amplification as described herein. As noted elsewhere herein,
the formation of the partial hairpin is generally desired to
prevent repriming of the replicated strand, e.g., making a copy of
a copy. As such, the partial hairpin is typically preferentially
formed from the amplification product during annealing as compared
to a primer annealing to the amplification product, e.g., the
hairpin will have a higher Tm than the primer product pair.
The second fragments are generally selected to be of a length that
is suitable for subsequent sequencing. For short read sequencing
technologies, such fragments will typically be from about 50 bases
to about 1000 bases in sequenceable length, from about 50 bases to
about 900 bases in sequenceable length, from about 50 bases to
about 800 bases in sequenceable length, from about 50 bases to
about 700 bases in sequenceable length, from about 50 bases to
about 600 bases in sequenceable length, from about 50 bases to
about 500 bases in sequenceable length, from about 50 bases to
about 400 bases in sequenceable length, from about 50 bases to
about 300 bases in sequenceable length, from about 50 bases to
about 250 bases in sequenceable length, from about 50 bases to
about 200 bases in sequenceable length, or from about 50 bases to
about 100 bases in sequenceable length, including the barcode
sequence segments, and functional sequences that are subjected to
the sequencing process.
Once the overlapping, barcoded second fragment sets are generated,
they may be pooled for subsequent processing and ultimately,
sequencing. For example, in some cases, the barcoded fragments may
be subsequently subjected to additional amplification, e.g., PCR
amplification, as described elsewhere herein. Likewise, these
fragments may additionally, or concurrently, be provided with
sample index sequences to identify the sample from which
collections of barcoded fragments have derived, as well as
providing additional functional sequences for use in sequencing
processes.
In addition, clean up steps may also optionally be performed, e.g.,
to purify nucleic acid components from other impurities, to size
select fragment sets for sequencing, or the like. Such clean up
steps may include purification and/or size selection upon SPRI
beads (such as Ampure.RTM. beads, available from Beckman Coulter,
Inc.). In some cases, multiple process steps may be carried out in
an integrated process while the fragments are associated with SPRI
beads, e.g., as described in Fisher et al., Genome Biol.
2011:12(1):R1 (E-pub Jan. 4, 2011), which is incorporated herein by
reference in its entirety for all purposes.
As noted previously, in many cases, short read sequencing
technologies are used to provide the sequence information for the
second fragment sets. Accordingly, in preferred aspects, second
fragment sets will typically comprise fragments that, when
including the barcode sequences, will be within the read length of
the sequencing system used. For example, for Illumina HiSeq.RTM.
sequencing, such fragments may be between generally range from
about 100 bases to about 200 bases in length, when carrying out
paired end sequencing. In some cases, longer second fragments may
be sequenced when accessing only the terminal portions of the
fragments by the sequencing process.
As will be appreciated, despite being based upon short sequence
data, one can infer that two sequences sharing the same barcode
likely originated from the same longer first fragment sequence,
especially where such sequences are otherwise assemble-able into a
contiguous sequence segment, e.g., using other overlapping
sequences bearing the common barcode. Once the first fragments are
assembled, they may be assembled into larger sequence segments,
e.g., the full length genetic component.
In one exemplary process, one or more fragments of one or more
template nucleic acid sequences may be barcoded using a method
described herein. A fragment of the one or more fragments may be
characterized based at least in part upon a nucleic acid barcode
sequence attached thereto. Characterization of the fragment may
also include mapping the fragment to its respective template
nucleic acid sequence or a genome from which the template nucleic
acid sequence was derived. Moreover, characterization may also
include identifying an individual nucleic acid barcode sequence and
a sequence of a fragment of a template nucleic acid sequence
attached thereto.
In some cases, sequencing methods described herein may be useful in
characterizing a nucleic acid segment or target nucleic acid. In
some example methods, a nucleic acid segment may be characterized
by co-partitioning the nucleic acid segment and a bead (e.g.,
including any suitable type of bead described herein) comprising a
plurality of oligonucleotides that include a common nucleic acid
barcode sequence, into a partition (including any suitable type of
partition described herein, such as, for example, a droplet). The
oligonucleotides may be releasably attached to the bead (e.g.,
releasable from the bead upon application of a stimulus to the
bead, such as, for example, a thermal stimulus, a photo stimulus,
and a chemical stimulus) as described elsewhere herein, and/or may
comprise one or more functional sequences (e.g., a primer sequence,
a primer annealing sequence, an immobilization sequence, any other
suitable functional sequence described elsewhere herein, etc.)
and/or one or more sequencing primer sequences as described
elsewhere herein. Moreover, any suitable number of oligonucleotides
may be attached to the bead, including numbers of oligonucleotides
attached to beads described elsewhere herein.
Within the partition, the oligonucleotides may be attached to
fragments of the nucleic segment or to copies of portions of the
nucleic acid segment, such that the fragments or copies are also
attached to the common nucleic barcode sequence. The fragments may
be overlapping fragments of the nucleic acid segment and may, for
example, provide greater than 2.times. coverage, greater than
5.times. coverage, greater than 10.times. coverage, greater than
20.times. coverage, greater than 40.times. coverage, or even
greater coverage of the nucleic acid segment. In some cases, the
oligonucleotides may comprise a primer sequence capable of
annealing with a portion of the nucleic acid segment or a
complement thereof. In some cases, the oligonucleotides may be
attached by extending the primer sequences of the oligonucleotides
to replicate at least a portion of the nucleic acid segment or
complement thereof, to produce a copy of at least a portion of the
nucleic acid segment comprising the oligonucleotide, and, thus, the
common nucleic acid barcode sequence.
Following attachment of the oligonucleotides to the fragments of
the nucleic acid segment or to the copies of the portions of the
nucleic acid segment, the fragments of the nucleic acid segment or
the copies of the portions of the nucleic acid segment and the
attached oligonucleotides (including the oligonucleotide's barcode
sequence) may be sequenced via any suitable sequencing method,
including any type of sequencing method described herein, to
provide a plurality of barcoded fragment sequences or barcoded copy
sequences. Following sequencing, the fragments of the nucleic acid
segment or the copies of the portions of the nucleic acid segment
can be characterized as being linked within the nucleic acid
segment at least in part, upon their attachment to the common
nucleic acid barcode sequence. As will be appreciated, such
characterization may include sequences that are characterized as
being linked and contiguous, as well as sequences that may be
linked within the same fragment, but not as contiguous sequences.
Moreover, the barcoded fragment sequences or barcoded copy
sequences generated during sequencing can be assembled into one or
more contiguous nucleic acid sequences based at least in part on
the common nucleic acid barcode sequence and/or a non-barcode
portion of the barcoded fragment sequences or barcoded copy
sequences.
In some cases, a plurality of nucleic acid segments (e.g.,
fragments of at least a portion of a genome, as described elsewhere
herein) may be co-partitioned with a plurality of different beads
in a plurality of separate partitions, such that each partition of
a plurality of different partitions of the separate partitions
contains a single bead. The plurality of different beads may
comprise a plurality of different barcode sequences (e.g., at least
1,000 different barcode sequences, at least 10,000 different
barcode sequences, at least 100,000 different barcode sequences, at
least 1,000,000 different barcodes sequences, or any other number
of different barcode sequences as described elsewhere herein). In
some cases, two or more, three or more, four or more, five or more,
six or more, seven or more of the plurality of separate partitions
may comprise beads that comprise the same barcode sequence. In some
cases, at least 0.01%, 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%
of the separate partitions may comprise beads having the same
barcode sequence. Moreover, each bead may comprise a plurality of
attached oligonucleotides that include a common nucleic acid
barcode sequence.
Following co-partitioning, barcode sequences can be attached to
fragments of the nucleic acid segments or to copies of portions of
the nucleic acid segments in each partition. The fragments of the
nucleic acid segments or the copies of the portions of the nucleic
acid segments can then be pooled from the separate partitions.
After pooling, the fragments of the nucleic acid segments or copies
of the portions of the nucleic acid segments and any associated
barcode sequences can be sequenced (e.g., using any suitable
sequencing method, including those described herein) to provide
sequenced fragment or sequenced copies. The sequenced fragments or
sequenced copies can be characterized as deriving from a common
nucleic acid segment, based at least in part upon the sequenced
fragments or sequenced copies comprising a common barcode sequence.
Moreover, sequences obtained from the sequenced fragments or
sequenced copies may be assembled to provide a contiguous sequence
of a sequence (e.g., at least a portion of a genome) from which the
sequenced fragments or sequenced copies originated. Sequence
assembly from the sequenced fragments or sequenced copies may be
completed based, at least in part, upon each of a nucleotide
sequence of the sequenced fragments and a common barcode sequence
of the sequenced fragments.
In another example method, a target nucleic acid may be
characterized by partitioning fragments of the target nucleic acid
into a plurality of droplets. Each droplet can comprise a bead
attached to a plurality of oligonucleotides comprising a common
barcode sequence. The common barcode sequence can be attached to
fragments of the fragments of the target nucleic acid in the
droplets. The droplets can then be pooled and the fragments and
associated barcode sequences of the pooled droplets sequenced using
any suitable sequencing method, including sequencing methods
described herein. Following sequencing, the fragments of the
fragments of the target nucleic acid may be mapped to the fragments
of the target nucleic acid based, at least in part, upon the
fragments of the fragments of the target nucleic acid comprising a
common barcode sequence.
The application of the methods, compositions and systems described
herein in sequencing may generally be applicable to any of a
variety of different sequencing technologies, including NGS
sequencing technologies such as Illumina MiSeq, HiSeq and X10
Sequencing systems, as well as sequencing systems available from
Life Technologies, Inc., such as the Ion Torrent line of sequencing
systems. While discussed in terms of barcode sequences, it will be
appreciated that the sequenced barcode sequences may not include
the entire barcode sequence that is included, e.g., accounting for
sequencing errors. As such, when referring to characterization of
two barcode sequences as being the same barcode sequence, it will
be appreciated that this may be based upon recognition of a
substantial portion of a barcode sequence, e.g., varying by fewer
than 5, 4, 3, 2 or even a single base.
Sequencing from Small Numbers of Cells
Methods provided herein may also be used to prepare polynucleotides
contained within cells in a manner that enables cell-specific
information to be obtained. The methods enable detection of genetic
variations from very small samples, such as from samples comprising
about 10-100 cells. In some cases, about 1, 5, 10, 20, 30, 40, 50,
60, 70, 80, 90 or 100 cells may be used in the methods described
herein. In some cases, at least about 1, 5, 10, 20, 30, 40, 50, 60,
70, 80, 90 or 100 cells may be used in the methods described
herein. In other cases, at most about 5, 10, 20, 30, 40, 50, 60,
70, 80, 90 or 100 cells may be used in the methods described
herein.
In an example, a method may comprise partitioning a cellular sample
(or crude cell extract) such that at most one cell (or extract of
one cell) is present within a partition, e.g., fluidic droplet, and
is co-partitioned with the barcode oligonucleotides, e.g., as
described above. Processing then involves lysing the cells,
fragmenting the polynucleotides contained within the cells,
attaching the fragmented polynucleotides to barcoded beads, pooling
the barcoded beads, and sequencing the resulting barcoded nucleic
acid fragments.
As described elsewhere herein, the barcodes and other reagents may
be encapsulated within, coated on, associated with, or dispersed
within a bead (e.g. gel bead). The bead may be loaded into a
fluidic droplet contemporaneously with loading of a sample (e.g. a
cell), such that each cell is contacted with a different bead. This
technique may be used to attach a unique barcode to
oligonucleotides obtained from each cell. The resulting tagged
oligonucleotides may then be pooled and sequenced, and the barcodes
may be used to trace the origin of the oligonucleotides. For
example, oligonucleotides with identical barcodes may be determined
to originate from the same cell, while oligonucleotides with
different barcodes may be determined to originate from different
cells.
The methods described herein may be used to detect a specific gene
mutation that may indicate the presence of a disease, such as
cancer. For example, detecting the presence of a V600 mutation in
the BRAF gene of a colon tissue sample may indicate the presence of
colon cancer. In other cases, prognostic applications may include
the detection of a mutation in a specific gene or genes that may
serve as increased risk factors for developing a specific disease.
For example, detecting the presence of a BRCA1 mutation in a
mammary tissue sample may indicate a higher level of risk to
developing breast cancer than a person without this mutation. In
some examples, this disclosure provides methods of identifying
mutations in two different oncogenes (e.g., KRAS and EGRF). If the
same cell comprises genes with both mutations, this may indicate a
more aggressive form of cancer. In contrast, if the mutations are
located in two different cells, this may indicate that the cancer
may be more benign, or less advanced.
Analysis of Gene Expression
Methods of the disclosure may be applicable to processing samples
for the detection of changes in gene expression. A sample may
comprise a cell, mRNA, or cDNA reverse transcribed from mRNA. The
sample may be a pooled sample, comprising extracts from several
different cells or tissues, or a sample comprising extracts from a
single cell or tissue.
Cells may be placed directly into a fluidic droplet and lysed.
After lysis, the methods of the disclosure may be used to fragment
and barcode the oligonucleotides of the cell for sequencing.
Oligonucleotides may also be extracted from cells prior to
introducing them into a fluidic droplet used in a method of the
disclosure. Reverse transcription of mRNA may be performed in a
fluidic droplet described herein, or outside of such a fluidic
droplet. Sequencing cDNA may provide an indication of the abundance
of a particular transcript in a particular cell over time, or after
exposure to a particular condition.
Partitioning Polynucleotides from Cells or Proteins
In one example the compositions, methods, devices, and kits
provided in this disclosure may be used to encapsulate cells or
proteins within the fluidic droplets. In one example, a single cell
or a plurality of cells (e.g., 2, 10, 50, 100, 1000, 10000, 25000,
50000, 10000, 50000, 1000000, or more cells) may be loaded onto,
into, or within a bead along with a lysis buffer within a fluidic
droplet and incubated for a specified period of time. The bead may
be porous, to allow washing of the contents of the bead, and
introduction of reagents into the bead, while maintaining the
polynucleotides of the one or more cells (e.g. chromosomes) within
the fluidic droplets. The encapsulated polynucleotides of the one
or more cells (e.g. chromosomes) may then be processed according to
any of the methods provided in this disclosure, or known in the
art. This method can also be applied to any other cellular
component, such as proteins.
Epigenetic Applications
Compositions, methods, devices, and kits of this disclosure may be
useful in epigenetic applications. For example, DNA methylation can
be in indicator of epigenetic inheritance, including single
nucleotide polymorphisms (SNPs). Accordingly, samples comprising
nucleic acid may be treated in order to determine bases that are
methylated during sequencing. In some cases, a sample comprising
nucleic acid to be barcoded may be split into two aliquots. One
aliquot of the sample may be treated with bisulfite in order to
convert unmethylated cytosine containing nucleotides to uracil
containing nucleotides. In some cases, bisulfite treatment can
occur prior to sample partitioning or may occur after sample
partitioning. Each aliquot may then be partitioned (if not already
partitioned), barcoded in the partitions, and additional sequences
added in bulk as described herein to generate sequencer-ready
products. Comparison of sequencing data obtained for each aliquot
(e.g., bisulfite-treated sample vs. untreated sample) can be used
to determine which bases in the sample nucleic acid are
methylated.
In some cases, one aliquot of a split sample may be treated with
methylation-sensitive restriction enzymes (MSREs). Methylation
specific enzymes can process sample nucleic acid such that the
sample nucleic acid is cleaved as methylation sites. Treatment of
the sample aliquot can occur prior to sample partitioning or may
occur after sample partitioning and each aliquot may be partitioned
used to generate barcoded, sequencer-ready products. Comparison of
sequencing data obtained for each aliquot (e.g., MSRE-treated
sample vs. untreated sample) can be used to determine which bases
in the sample nucleic acid are methylated.
Low Input DNA Applications
Compositions and methods described herein may be useful in the
analysis and sequencing of low polynucleotide input applications.
Methods described herein, such as PHASE, may aid in obtaining good
data quality in low polynucleotide input applications and/or aid in
filtering out amplification errors. These low input DNA
applications include the analysis of samples to sequence and
identify a particular nucleic acid sequence of interest in a
mixture of irrelevant or less relevant nucleic acids in which the
sequence of interest is only a minority component, to be able to
individually sequence and identify multiple different nucleic acids
that are present in an aggregation of different nucleic acids, as
well as analyses in which the sheer amount of input DNA is
extremely low. Specific examples include the sequencing and
identification of somatic mutations from tissue samples, or from
circulating cells, where the vast majority of the sample will be
contributed by normal healthy cells, while a small minority may
derive from tumor or other cancer cells. Other examples include the
characterization of multiple individual population components,
e.g., in microbiome analysis applications, where the contributions
of individual population members may not otherwise be readily
identified amidst a large and diverse population of microbial
elements. In a further example, being able to individually sequence
and identify different strands of the same region from different
chromosomes, e.g., maternal and paternal chromosomes, allows for
the identification of unique variants on each chromosome.
Additional examples of low polynucleotide input applications of the
compositions, methods, and systems described herein are set forth
in U.S. Provisional Patent Application No. 62/017,580, filed Jun.
26, 2014.
The advantages of the methods and systems described herein are
clearer upon a discussion of the problems confronted in the present
state of the art. In analyzing the genetic makeup of sample
materials, e.g., cell or tissue samples, most sequencing
technologies rely upon the broad amplification of target nucleic
acids in a sample in order to create enough material for the
sequencing process. Unfortunately, during these amplification
processes, majority present materials will preferentially overwhelm
portions of the samples that are present at lower levels. For
example, where a genetic material from a sample is comprised of 95%
normal tissue DNA, and 5% of DNA from tumor cells, typical
amplification processes, e.g., PCR based amplification, will
quickly amplify the majority present material to the exclusion of
the minority present material. Furthermore, because these
amplification reactions are typically carried out in a pooled
context, the origin of an amplified sequence, in terms of the
specific chromosome, polynucleotide or organism will typically not
be preserved during the process.
In contrast, the methods and systems described herein partition
individual or small numbers of nucleic acids into separate reaction
volumes, e.g., in droplets, in which those nucleic acid components
may be initially amplified. During this initial amplification, a
unique identifier may be coupled to the components to the
components that are in those separate reaction volumes. Separate,
partitioned amplification of the different components, as well as
application of a unique identifier, e.g., a barcode sequence,
allows for the preservation of the contributions of each sample
component, as well as attribution of its origin, through the
sequencing process, including subsequent amplification processes,
e.g., PCR amplification.
The term "about," as used herein and throughout the disclosure,
generally refers to a range that may be 15% greater than or 15%
less than the stated numerical value within the context of the
particular usage. For example, "about 10" would include a range
from 8.5 to 11.5.
As will be appreciated, the instant disclosure provides for the use
of any of the compositions, libraries, methods, devices, and kits
described herein for a particular use or purpose, including the
various applications, uses, and purposes described herein. For
example, the disclosure provides for the use of the compositions,
methods, libraries, devices, and kits described herein in
partitioning species, in partitioning oligonucleotides, in
stimulus-selective release of species from partitions, in
performing reactions (e.g., ligation and amplification reactions)
in partitions, in performing nucleic acid synthesis reactions, in
barcoding nucleic acid, in preparing polynucleotides for
sequencing, in sequencing polynucleotides, in polynucleotide
phasing (see e.g., U.S. Provisional Patent Application No.
62/017,808, filed Jun. 26, 2014), in sequencing polynucleotides
from small numbers of cells, in analyzing gene expression, in
partitioning polynucleotides from cells, in mutation detection, in
neurologic disorder diagnostics, in diabetes diagnostics, in fetal
aneuploidy diagnostics, in cancer mutation detection and forensics,
in disease detection, in medical diagnostics, in low input nucleic
acid applications, such as circulating tumor cell (CTC) sequencing,
in a combination thereof, and in any other application, method,
process or use described herein.
Any concentration values provided herein are provided as admixture
concentration values, without regard to any in situ conversion,
modification, reaction, sequestration or the like. Moreover, where
appropriate, the sensitivity and/or specificity of methods (e.g.,
sequencing methods, barcoding methods, amplification methods,
targeted amplification methods, methods of analyzing barcoded
samples, etc.) described herein may vary. For example, a method
described herein may have specificity of greater than 50%, 70%,
75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 99.5% and/or a sensitivity of greater than
50%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.
Additional Sequencing Approaches
A wide variety of different sequencing technologies are practiced
across broad ranging industries, including biotechnology,
pharmaceutical research, medical diagnostics, agriculture, basic
research, food safety and so on. These technologies include the
older Sanger sequencing methods where nested fragments of template
nucleic acids terminated with the four different nucleotides
bearing distinguishable labels are separated by their size and
identified as to their terminating nucleotide by the
distinguishable label.
Sequencing methods also include more recent "sequencing by
synthesis", or SBS, methods where the iterative addition of
specific nucleotides in a template dependent, polymerase mediated
extension reaction are identified and used to provide the
underlying sequence of the template nucleic acid. These SBS
processes are generally divided into (1) short read sequencing
technologies, e.g., employed in Illumina HiSeq, MiSeq, and NextSeq
sequencing systems, as well as the Ion Torrent Proton and PGM
systems, available from Thermo Fisher, and (2) long read sequencing
technologies such as single molecule, real time, or SMRT.RTM.
sequencing systems available from Pacific Biosciences.
The short read technologies generally utilize an ensemble approach
where patches or clusters of identical nucleic acid template
molecules arrayed on substrates are observed or detected in
separate cycles of nucleotide addition, in order to identify the
added bases in a stepwise fashion. By providing large numbers of
clusters each representing different molecules, one can sequence
large numbers of different nucleic acid fragments during a
sequencing run. Further, by relying upon the consensus of the
identified base added over all of the molecules within a given
cluster, i.e., having hundreds of thousands of molecules, any low
level inaccuracy of the extension reaction, e.g., incorporating an
incorrect base, is overwhelmed by the correct base addition,
leading to very high accuracy rates for sequence reads. However,
because of inherent inefficiencies in the extension reactions,
extension of the various template molecules within any given
cluster can, over time, go out of phase with one another, resulting
in an inability to accurately call bases after a few hundred bases
of read length, even in an ensemble approach.
By contrast, the long read, single molecule SBS methods, such as
SMRT sequencing, detect individual bases within a single nucleic
acid molecule. SMRT sequencing, for example, relies upon the
observation of incorporation of individual bases in a replication
of a template molecule, as the template is being replicated by a
single DNA polymerase enzyme, where the sequential addition of
bases to the duplicating strand are observable using special
optical detection techniques and fluorophore labeled nucleotides.
By observing replication of a single long nucleic acid template
molecule, one can obtain very long read lengths, e.g., on the order
of 10s of thousands of bases. However, as these techniques observe
replication of a single nucleic acid molecule, any mistakes made in
the polymerase reaction are observed and incorporated into the
perceived read. Furthermore, in order to avoid confounding sequence
information, highly accurate polymerases, e.g., that possess
proofreading capabilities, are not used. This results in single
pass accuracies of only on the order of 85% of base calls being
correct. Remedies for this deficiency in single pass accuracy
employ the template molecules in a circular structure, such that
multiple passes by the single polymerase around the circular
molecule may be made, mimicking an ensemble approach to improving
accuracy, e.g., multiple sequencing passes over the same molecule
of sequence provide a higher consensus accuracy for that
sequence.
In still other approaches, individual template molecules would be
directly read out as the molecule itself passes through a detecting
zone, e.g., in a nanopore sequencing system. Again, while these
systems have been described in proof of principle experiments, they
are generally not commercially available, and are generally prone
to inaccuracy and production of noisy data.
For most of these sequencing technologies, there are significant
steps that are taken up front of the actual sequencing process, in
order to provide template nucleic acids in a sequenceable format
for the sequencing system being used. These involve conventional
process steps of purifying the nucleic acids to be sequenced away
from other material in a sample, e.g., extracting it from cells or
tissue, purifying away contaminating proteins, enzymes and other
cellular debris, as well as steps of incorporating operable
components onto the nucleic acids in order to allow for sequencing,
such as primer sequences, adapter sequences, hairpin sequences and
identifier sequences, such as oligonucleotide barcodes or sample
index oligonucleotides. A number of different process steps have
evolved for preparing sequenceable libraries of nucleic acid
molecules (also termed "sequencing libraries" herein), many of
which are highly dependent upon the sequencing system being
used.
Additional Barcoding Libraries
In one example, a partitioning and barcoding process is used to
derive long-range sequence information from template nucleic acids
without the need for long read sequencing processes. In brief, long
fragments of nucleic acids from sample, e.g., cells or tissue, are
partitioned into discrete aqueous droplets in an aqueous:oil
emulsion. Beads bearing populations of barcoded primer sequences
are co-partitioned into these droplets along with the sample
fragments, polymerization reaction components, e.g., polymerase
enzyme, nucleoside triphosphates, Mg2+, and the like. The barcoded
primers are released from the beads and allowed to prime along
portions of the template nucleic acids to produce replicate
fragments of the template. As a result, each partition or droplet
can include replicate fragments of the original starting fragments,
but where each fragment includes a barcode sequence that is
attributable to the single bead partitioned into a given droplet.
These replicate fragments are then further processed, e.g., to
attach additional functional sequences, such as amplification
primer sequences, other sequencer specific sequences, e.g., flow
cell attachment sequences, sequencing primer sequences, and the
like, as well as to amplify the number of fragments in order to put
them through the sequencing processes.
Sequencing of the replicated, barcoded fragments then yields short
sequence reads that also include a barcode sequence. This barcode
sequence can then be used, along with sequence information, to
attribute the associated fragment sequence to an originating
starting fragment, thereby providing long range sequence
information, e.g., as to the originating long fragment, from short
read sequences. By ensuring that replicate fragments cover the
entire originating fragment, even multiple times, one can readily
assemble the sequence into virtual long reads of the originating
fragment. In addition, even without complete multifold coverage
used for complete de novo sequencing, the presence of common
barcodes on different short sequences can allow the inference of
longer range linkage between the two different short sequences,
providing numerous advantages over short read sequencing alone,
e.g., in genome mapping, structural variant detection,
identification of phased variants (see, e.g., U.S. Patent
Application No. 62/072,214, filed Oct. 29, 2014, which is
incorporated herein by reference in its entirety for all purposes),
as well as other valuable long range sequence linkage information.
These methods and their applications are discussed in detail in,
for example, co-pending U.S. patent application Ser. No.
14/316,383, filed Jun. 26, 2014, 62/017,808, filed Jun. 26, 2014,
62/072,214, filed Oct. 29, 2014, 62/072,164, filed Oct. 29, 2014,
and 62/017,558, filed Jun. 26, 2014, the full disclosures of which
are each incorporated herein by reference in their entireties for
all purposes.
Additional Fragmentation and Barcoding
As described herein, provided are methods, and systems for
preparing improved sequencing libraries from sample nucleic acids.
The improved sequencing libraries provide one or more of more
uniform coverage, lower sequence error rates, higher amplification
rates of the original sequence, and lower chimera generation
rates.
As noted above, a method for providing barcoded replicate fragments
of template nucleic acids to use as a sequencing library is
described in detail in co-pending U.S. patent application Ser. No.
14/316,383, filed Jun. 26, 2014, and previously incorporated herein
by reference. Briefly, and as shown in FIG. 19A-F, oligonucleotides
that include a barcode sequence are co-partitioned in, e.g., a
droplet 102 in an emulsion, along with a sample nucleic acid 104.
The oligonucleotides 108 may be provided on a bead 106 that is
co-partitioned with the sample nucleic acid 104, which
oligonucleotides are preferably releasable from the bead 106, as
shown in FIG. 19A. The oligonucleotides 108 include a barcode
sequence 112, in addition to one or more functional sequences,
e.g., sequences 110, 114 and 116. For example, oligonucleotide 108
is shown as comprising barcode sequence 112, as well as sequence
110 that may function as an attachment or immobilization sequence
for a given sequencing system, e.g., a P5 sequence used for
attachment in flow cells of an Illumina Hiseq or Miseq system. As
shown, the oligonucleotides also include a primer sequence 116,
which may include a universal, random or targeted N-mer for priming
replication of portions of the sample nucleic acid 104. Also
included within oligonucleotide 108 is a sequence 114 which may
provide a sequencing priming region, such as a "read1" or R1
priming region, that is used to prime polymerase mediated, template
directed sequencing by synthesis reactions in sequencing systems.
In many cases, the barcode sequence 112, immobilization sequence
110 and R1 sequence 114 may be common to all of the
oligonucleotides attached to a given bead. The primer sequence 116
may vary for random N-mer primers, or may be common to the
oligonucleotides on a given bead for certain targeted applications.
Although described with reference to the specific positioning and
type of functional sequence segment elements within the barcode
oligonucleotides, it will be appreciated that the position and
nature of the functional segments within a barcode oligonucleotide
may vary. For example, primer sequences for different sequencing
systems may be employed in place of the P5, read1, etc. primers.
Likewise, as noted elsewhere herein, targeted primer sequences may
be provided to permit attachment of barcode sequences to targeted
portions of a genome or sample genetic material. Additionally, in
some cases, the positional context of the different segments may be
changed. For example, in some cases, it may be desirable to
position the barcode sequence segment 5' of the sequence read
primer or R1 segment 114, e.g., between segments 114 and 116, so
that the barcode can be sequenced in a first pass or initial
sequence read, e.g., following priming of the read1 sequence during
the sequencing of the resultant barcoded fragments, as opposed to
obtaining the barcode read on a subsequent sequencing read of a
reverse complement. This and a variety of other variations are
envisioned by the present disclosure.
Based upon the presence of primer sequence 116, the
oligonucleotides are able to prime the sample nucleic acid as shown
in FIG. 19B, which allows for extension of the oligonucleotides 108
and 108a using polymerase enzymes and other extension reagents also
co-partitioned with the bead 106 and sample nucleic acid 104. As
described elsewhere herein, these polymerase enzymes may include
thermostable polymerases, e.g., where initial denaturation of
double stranded sample nucleic acids within the partitions is
desired. Alternatively, denaturation of sample nucleic acids may
precede partitioning, such that single stranded target nucleic
acids are deposited into the partitions, allowing the use of
non-thermostable polymerase enzymes, e.g., Klenow, phi29, Pol1, and
the like, where desirable. As shown in FIG. 19C, following
extension of the oligonucleotides that, for random N-mer primers,
can anneal to multiple different regions of the sample nucleic acid
104; multiple overlapping complements or fragments of the nucleic
acid are created, e.g., fragments 118 and 120. Although including
sequence portions that are complementary to portions of sample
nucleic acid, e.g., sequences 122 and 124 (also referred to as
"inserts"), these constructs are generally referred to herein as
comprising fragments of the sample nucleic acid 104, having the
attached barcode sequences. In some cases, it may be desirable to
artificially limit the size of the replicate fragments that are
produced in order to maintain manageable fragment sizes from the
first amplification steps. In some cases, this may be accomplished
by mechanical means, as described above, e.g., using fragmentation
systems like a Covaris system, or it may be accomplished by
incorporating random extension terminators, e.g., at low
concentrations, to prevent the formation of excessively long
fragments.
These fragments may then be subjected to sequence analysis, or they
may be subjected to further processing, e.g., to amplify the amount
of nucleic acids available for sequencing, e.g., as shown in the
process illustrated in FIG. 19D and/or provide additional
functional sequences. For example, additional oligonucleotides,
e.g., oligonucleotide 108b, also released from bead 106, may prime
the fragments 118 and 120. In particular, again, based upon the
presence of the random N-mer primer 116b in oligonucleotide 108b
(which in many cases can be different from other random N-mers in a
given partition, e.g., primer sequence 116), the oligonucleotide
anneals with the fragment 118, and is extended to create a
complement 126 to at least a portion of fragment 118 which includes
sequence 128, that comprises a duplicate of a portion of the sample
nucleic acid sequence. Extension of the oligonucleotide 108b
continues until it has replicated through the oligonucleotide
portion 108 of fragment 118. As illustrated in FIG. 19D, the
oligonucleotides may be configured to prompt a stop in the
replication by the polymerase at a desired point, e.g., after
replicating through sequences 116 and 114 of oligonucleotide 108
that is included within fragment 118. In some cases, this is
achieved through the incorporation of nucleotide or nucleotide
analogues that are not processed by the polymerase being used for
the replication reaction. For example, in many cases, uracil
containing bases may be included in the primer sequences to stop
replication by a polymerase that does not read through uracil
containing bases. This may be done in order to provide for the
generation of partial hairpin sequences, e.g., that have partial
internal complementarity, in order to prevent excessive replication
of copies and the associated bias, e.g., partial hairpins would be
removed, at least in part, from subsequent replication steps.
As described herein, this may be accomplished by different methods,
including, for example, the incorporation of different nucleotides
and/or nucleotide analogues that are not capable of being processed
by the polymerase enzyme used. For example, this may include the
inclusion of uracil containing nucleotides within the sequence
region 112 to cause a non-uracil tolerant polymerase to cease
replication of that region. As a result, a fragment 126 is created
that includes the full-length oligonucleotide 108b at one end,
including the barcode sequence 112, the attachment sequence 110,
the R1 primer region 114, and the random n-mer sequence 116b.
At the other end of the sequence can be included the complement
116' to the random n-mer of the first oligonucleotide 108, as well
as a complement to all or a portion of the R1 sequence, shown as
sequence 114'. The R1 sequence 114 and its complement 114' are then
able to hybridize together to form a partial hairpin structure 128.
As will be appreciated because the random-n-mers differ among
different oligonucleotides, these sequences and their complements
generally would not be expected to participate in hairpin
formation, e.g., sequence 116', which is the complement to random
N-mer 116, would generally not be expected to be complementary to
random n-mer sequence 116b. This generally would not be the case
for other applications, e.g., targeted primers, where the N-mers
may be common among oligonucleotides within a given partition.
By forming these partial hairpin structures, it allows for the
removal of a large number of first level duplicates of the sample
sequence from further replication, e.g., reducing the prevalence of
iterative copying of copies. The partial hairpin structure also
provides a useful structure for subsequent processing of the
created fragments, e.g., fragment 126. Additionally, the use of
U-containing oligonucleotides and non-U processing polymerases in
the barcoding process reduces the amount of primer-dimer artifacts
during that barcoding process (e.g., as little or no extension
would occur across a U-containing primer that is serving as a
template for extension), that would otherwise reduce the efficiency
of the process.
In one example of an improved approach, a partitioning method as
described above is employed, but with a separate primer
oligonucleotide added to the reaction mix that also includes
sufficient functional sequence elements to be able to permit
barcode attachment, but which not part of the barcode
oligonucleotide. This approach is schematically illustrated in FIG.
20A. As shown, a bead 206 bearing the barcode oligonucleotide 208
to be co-partitioned with the sample nucleic acid fragment includes
oligonucleotides that have a barcode sequence as well as one or
more additional sequences, e.g., attachment sequence 210 (e.g.,
P5), barcode sequence 212, and sequencing primer sequence 214
(e.g., R1). As noted above, the barcode portion 212 of the sequence
can vary among different beads, while at least one of the
additional sequences is constant across the various different
beads. In the example shown, the oligonucleotides 208 on the bead
206 include a variable barcode portion 212 and one or more constant
portions, which, as shown include, e.g., attachment sequence 210
and sequencing primer segment 214. Also co-partitioned with the
barcode oligonucleotides is a separate primer oligonucleotide 216
that includes a primer sequence portion 216a as well as a portion
216b that is identical to at least a portion of the constant
portion, e.g., sequencing primer 214, of barcode oligonucleotide
208. While primer sequence portion 216a is illustrated as a random
N-mer primer, it will again be appreciated that specific primer
sequences could also be employed, e.g., targeting specific priming
sequences or sequences adjacent to regions of interest in the
genome, for use in generating sequencing libraries for targeted
genes, gene panels, or portions of the genome, or primer sequences
that are less than completely random, e.g., as described elsewhere
herein.
Once co-partitioned along with the template nucleic acid 204, the
primer sequence portion 216a can anneal to portions of the template
204, and be extended to create replicate fragments 222 of the
template 204 that include both the priming sequence 216a and the
additional sequence segment 216b that is identical to at least a
portion of a constant portion, e.g., sequence 214, of barcode
oligonucleotide 208. Following the initial extension, a second
primer sequence 216 anneals to the newly created replicate fragment
222, and is extended to create a complementary replicate fragment
224 that includes sequence portion 226 that is complementary to at
least a portion of constant sequence segment 214, e.g., at the 5'
terminus) on barcode oligonucleotide 208 (as well as a complement
to the original primer sequence--shown as nnnn). The barcode
oligonucleotide is then able to anneal to the complementary
sequence portion 226 through constant segment 214, and extension of
that sequence results in a replicate copy 228 of the sample nucleic
acid sequence with an attached barcode sequence 212, as well as the
attached constant portions, e.g., attachment sequence 210 and
sequencing primer sequence 214, and a complementary sequence 230 to
the partial constant sequence 216b. As shown, both the barcode
oligonucleotide 208 and replicate fragment 224 are extended to
yield both replicate copy 228, and its complement 228c. As will be
appreciated, in some cases, the 5' terminus of the barcode
oligonucleotide may be provided with a blocking group to prevent
extension, e.g., preventing the generation of fragment 228, and
only allowing replication of the barcode oligonucleotide onto
fragment 224. This may be done in some instances in order to avoid
the barcode oligonucleotide priming in a less controlled fashion
against the underlying sample nucleic acids, e.g., the genome,
which could result in suboptimal library generation. A variety of
blocking groups or other non-extendible nucleotide groups may be
employed, including blocked nucleic acids, dideoxy terminated
nucleic acids, and the like.
Use of a separate primer sequence with the ability to attach
barcode sequences to it, in process, can provide advantages of
controllability to the priming operation that is separate from the
barcode library itself. In particular, a barcode library may be
constructed that is universally applicable for different
applications, where those different applications may benefit from
different priming strategies, e.g., other than purely random n-mer
priming. The application specific primer sequences may then be
added to the reaction mix, rather than having to reconstruct an
entire barcode library including primer sequences, to pursue the
desired application. In particular, one could readily substitute
targeted primer sequences, biased primer sequences, e.g., GC
biased, AT biased, or other structured primer sequences, e.g.,
having defined sub-motifs, sub-biases as to segments of the primer
sequence, etc., in order to optimize the library generation process
to the given application.
As discussed in greater detail below, additional processing steps
may be carried out on barcoded replicate nucleic acid fragments,
e.g., fragments 228 and 228c shown in FIG. 20A, in order to provide
additional functional sequences on those replicate fragments or
copies or complements of those fragments. For example, in some
cases as described below, additional amplification steps can be
carried out that couple additional functional sequences used for
sequencing processes, onto the end of the barcoded fragment, e.g.,
at end 230 of barcoded fragment 228. However, in certain aspects,
the attachment of additional sequences may be incorporated into the
barcoding replication process so as to yield fragments that include
both the barcode oligonucleotide portion and other functional
sequences at the opposing end of the replicate fragment. By way of
example, one may include, within the original barcoding reaction
mixture, a second set of primer sequences that include a priming
sequence, e.g., a random n-mer primer sequence that is coupled to
the desired additional functional sequences, e.g., the R2 and P7
sequences discussed elsewhere herein, allowing for a single step
reaction process for both barcoding a fragment at one end, and
attaching additional functional sequences at the other end. The
presence of functional sequences on both ends of the barcoded
fragments can then allow facile further processing of the
fragments. For example one may use these functional sequences in
the anteparallel amplification of the barcoded fragments.
This is schematically illustrated in FIG. 20B, and with reference
to FIG. 20A where second primer oligonucleotide 250 is introduced
into the reaction mixture along with the barcode oligonucleotides
208 and template 204. Second primer set 250 includes the additional
desired functional sequences 250b and 250c, which may be a read2
priming sequence and a P7 attachment sequence, respectively, in
addition to the primer sequence, e.g., random n-mer 250a.
Again, as with the process shown in FIG. 20A, first primer set 216
anneals to the template and extends along a portion of the template
204 to produce a first replicate fragment 222. The second primer
set 250 then anneals to replicate fragment 222 and extends along
that replicate fragment to produce a complementary copy 252 that
includes those functional sequence elements 250b and 250c, as well
as a complement to at least a portion of segment 214 on the barcode
oligonucleotide 208. The barcode oligonucleotide 208 can then
anneal to replicate fragment 252, where extension of the barcode
oligonucleotide (and fragment 252), can produce a barcoded
replicate fragment 254 and its complement 254c, both of which can
include the sequence segments included in the barcode
oligonucleotide or their complements, as well as those additional
functional sequences delivered by the second primer set 250, or
their complements. As will be appreciated, the presence of first
and second primer sets in the same reaction mixture can potentially
result in a set of replicate fragments that includes a number of
structures, including the desired structures, where the insert
segment is flanked on one side by the first primer set or its
complement and on the other side by the second primer set or its
complement. However, other arrangements can also be present,
including those where only one of either of the first or the second
primer sets flank both sides of an insert segment. In general, this
could be resolvable during a sequencing process, or by a subsequent
amplification process in which only sequences carrying both ends of
the desired sequence are present are amplified, e.g., using P5 and
P7 as the amplification primer sequences. For example, with respect
to replicate fragment 254c, one could selectively amplify this
segment by priming against the P7 sequence represented by segment
250c, while priming against the complement to the P5 sequence
segment (e.g., segment 210), as represented by segment 210c.
As will be appreciated, this simplified process described in FIG.
20B, may also be applied in a modified version of the process shown
in FIGS. 19A-F. In particular, two different primer sets may be
presented in the barcoding reaction mix in order to provide a "one
pot" reaction that results in barcoded fragments having functional
sequences at both ends.
This is schematically illustrated in FIG. 20C. As shown, a template
nucleic acid sequence 280, is co-partitioned along with a
barcode/primer oligonucleotide 260 and a second adapter/primer
sequence 270. The barcode/primer 260 is preferably partitioned,
releasably attached to a bead, and as a member of a diverse barcode
library, e.g., as described above. Adapter/primer sequence 270, as
it can typically include defined or common functional sequences,
may be partitioned in bulk, e.g., along with the nucleic acid
template 280, or other reagents added to the partitioning process,
e.g., enzymes, nucleotides, etc. In some cases, however, the
adapter/primer 270 may be partitioned releasably attached to the
same or a different bead from the barcode/primer 260.
Each of the barcode/primer 260 and adapter/primer 270 may include
additional functional sequences, in addition to the barcode and
primer portions. For example, barcode/primer sequence 260 is shown
as including barcode sequence 264, and a random n-mer primer
sequence 268, but also includes one or more additional functional
sequences, such as a flow cell attachment sequence, sequencing read
primer sequence, and the like. For ease of discussion, the example
illustrated in FIG. 20C is described where barcode primer 260
includes a P5 attachment sequence 262, a barcode sequence 264, a
first sequence read primer, e.g., a read1 primer sequence used in
Illumina sequencing processes, and a random sample priming sequence
or n-mer 268. The adapter primer 270 is described in terms of
including a P7 attachment sequence 272, e.g., as used in Illumina
sequencing, a second sequence read primer, e.g., Read2 primer 274,
and a random priming sequence or n-mer 276.
Upon initiation of a primer extension reaction, e.g., upon one or
more of mixing the requisite reagents, release of the barcode
primer from the beads and/or commencement of thermal cycling of the
reaction mixture, the primer sequences, e.g., 268 and 276, can
anneal with the template nucleic acid 280 (only shown as primer 268
annealing), and be extended along the template creating a
replicated portion of the template that is attached to the
barcode/primer as extension product 282. Although not shown, along
with extension product 282, extension products can be created based
upon extension of adapter/primer 270 that has annealed to the
template sequence.
Following this first extension, the extension product then serves
as a template for subsequent rounds of primer annealing and
extension. As shown, adapter/primer 270 anneals to extension
product 282, and is extended to replicate the portion of the
extension product 282 that includes a complementary portion to the
original template sequence (shown as insert segment 284), and the
original barcode/primer, to create extension product 286, that
includes a complement to the original barcode primer, shown as
segment 260c. Again, although not shown, a similar complementary
reaction can be carried out to replicate the extension products
created from extension of the adapter/primer sequence along the
template, which could result in the barcode primer at one end of an
insert sequence, and the complement of the adapter/primer sequence
at the other end of the insert.
As will be appreciated, and as alluded to above, in some cases, the
same sequence or its complement could be present on both ends of an
insert in roughly 50% of the extension products. Conveniently,
however, the products of the barcoding and adapter attachment
processes described above, e.g., including extension product 286,
and those `products` that have the same sequence or its complement
on each end, may be subjected to additional processing. In
particular, in at least one example, the products may be subjected
to anteparallel amplification by priming against both of the P5 and
P7 sequences using a PCR process. As a result, those fragments that
include both the P5 and P7 sequences, or their complements can be
rapidly, and exponentially amplified, which the other `products`
will not.
As will be appreciated, specific reference to the functional
sequences and their complements in this example is illustrative,
and not limiting. In practice, a particular sequence or its
complement, may be chosen for any of the sequence segments
designated above, e.g., P5, P7, read1, read2, etc., depending upon
the desired end state of the desired products.
As will be appreciated, in some cases, the process of generating
barcoded replicate fragments from a long template nucleic acid can
have variations in the amount of coverage of the underlying nucleic
acid fragment, e.g., some areas being represented by more replicate
fragments than others, and that variation in coverage can translate
into the sequencing coverage for that template. Generally, it is
desirable to generate replicate fragments that represent more even
coverage over the full length of the template nucleic acid, or meet
a minimum coverage threshold as to significant portions of the
template sequence.
As alluded to above, in some cases, the make up of the primer
portion of the oligonucleotide, e.g., primer segment 116 of the
barcode oligonucleotide shown in FIG. 19A, or a primer segment 216
shown in FIG. 20A and FIG. 20B, may be adjusted to enhance library
preparation. In particular, in some cases, the make up of the
primer sequence used to anneal to the template nucleic acid can be
controlled in order to provide for more uniform sampling of the
template sequence, and as a result, more even sequence coverage. In
particular, by controlling the relative GC content of the primer
sequence, whether it is a random primer sequence or a more targeted
primer sequence, one can enhance the resulting sequencing coverage.
In some aspects the primer sequences are provided with greater than
a 50% GC content, preferably, greater than 60% GC content, greater
than 70% GC content or even 80% GC content or greater. In preferred
aspects, the GC content of the primer may be from 50% to about 90%
and any range defined thereby, or from about 50% to about 60%, from
about 60% to about 70%, from about 70% to about 80%, or from about
80% to about 90%.
In some cases, blends of primer subpopulations, each having a
different GC percentage may be employed, e.g., where the primers
contained in the overall mix have a range of GC concentrations from
greater than 50% to 90% or greater. In many cases, the primers can
range from greater than 50% GC up to about 80% GC. These primer
populations may span the entire range of GC concentrations in the
stated range, or they may constitute set subpopulations of primers
each having a distinct GC percentage.
For example, in some cases, subpopulations of primers may be
blended to create mixtures having set subpopulations of GC
concentrations in the primers, e.g., a primer subpopulation that
has 60% GC blended with a primer subpopulation that has 80% GC. As
will be appreciated, in such cases, the blends may include two,
three, four or more different subpopulations of primer constructs,
e.g., having differing GC content. Typically, such subpopulations
may be from 50% GC to 90% GC, while each subpopulation may be from
1% to 99% of the blend. In preferred aspects, the subpopulations
may have a GC content of between about 50% and 80% GC, inclusive,
and each subpopulation can make up from 10% to 90% of the total
primer population, from 20% to 80%, 30% to 70%, 40% to 60%, or even
50% for each subpopulation.
In addition to the above-described processes for improving library
preparation, one may also utilize modifications to the polymerase
reactions in order to provide improved libraries, e.g., with more
even coverage, lower error and lower chimera formation. In
particular, in at least one example, one may utilize different
polymerases in combination, in order to improve the reaction
products. In particular, by using polymerases that have different
but complementary properties, one can produce higher quality
libraries. By way of example, a blend of a first polymerase that
provides very low error rates in replicating template sequence
fragments, and a second polymerase that provides more even coverage
or higher reaction rate or greater processivity, can provide a
reaction that provides improved libraries. In one specific example,
a blend of a highly accurate and processive polymerase such as the
9.degree. North polymerase, retaining its wild type exonuclease
activity (exo+) may be blended with another archeal polymerase such
as Deep Vent polymerase, available from NEB provides sequencing
libraries having more uniform coverage and lower error rates than
either polymerase used alone.
FIG. 21 shows comparison of chimera and Q35 error rates of
different polymerase enzymes. As shown, the 9.degree. N (exo+)
polymerase demonstrates a relatively low Q35 error rate, but a
relatively high chimera rate when used on its own (see circle A).
In contrast, the Deep Vent polymerase illustrates a relatively
higher error rate, but a relatively lower chimera rate (See circle
B). When both enzymes are used in a blend of both enzymes, benefits
are seen over either alone in both chimera rate and error rate (See
circle C).
In addition to the processing described above, the methods
described herein may also be used for selective barcoding of
targeted genomic libraries. One approach for barcoding targeted
genomic libraries, e.g., sequencing libraries that include targeted
genetic regions, e.g., genes, gene panels, exomes, kinomes, etc.,
using the barcoding methods alluded to herein are described in
Provisional U.S. Patent Application No. 62/073,659, filed Oct. 31,
2014, and incorporated herein by reference in its entirety for all
purposes. In particular, the methods described utilize the
barcoding approaches described herein in order to attach barcodes
to genome (or sample) wide fragments, in order to provide an
indicator of original molecular context or attribution. Once the
fragments are barcoded, they may be selected for using conventional
targeting processes, e.g., pull-downs, e.g., using conventional
kits, e.g., pull down panels, exome kits etc., such as the
SureSelect.RTM. exome kits available from Agilent Technologies,
Inc. In an alternative approach, the barcodes may be attached to
the targeting sequences (also referred to as target baits or
targeted primers) using the methods described herein and
illustrated with reference to FIG. 24, which are then used to
create the targeted sequencing libraries that include the barcode
sequences, e.g., using process steps described herein. As will be
appreciated, although described as attaching the barcode sequences
to targeted primers, the methods described may be used in attaching
the barcode oligonucleotides to virtually any sequence, e.g., any
targeted, random, universal, or other primer sequence or probe,
without the need to incorporate a sample priming sequence, e.g., a
radon n-mer or targeted primer, on the barcode oligonucleotide on
the bead. In one example, a barcoded bead library, as described
above, is used to deliver a population of common barcode sequences
to an individual partition, e.g., as a droplet in an emulsion. The
bead may be co-partitioned along with a sample nucleic acid as
described above. Additionally, the bead can be co-partitioned with
a targeted primer sequence, e.g., a sequence that is the same as or
complementary to a specific targeted sequence of interest. The
targeted primer sequence can typically include a portion that
allows it to hybridize to a downstream portion of the barcode
oligonucleotide, in order for the barcoded primer to be extended
along the barcode oligonucleotide, thus replicating the barcode
into the targeted primer sequence. Replication of the now barcoded
targeting sequence can create a barcoded, targeted primer sequence
that can interrogate the sample nucleic acid for the targeted
region, and produce replicate fragments that include the barcode
sequence.
An example of this process is schematically illustrated in FIG. 24.
As shown, a barcoded bead from a barcode bead library as described
elsewhere herein, is provided with a barcode containing
oligonucleotide 602, that includes a barcode segment 604 along with
additional functional sequences, e.g., an attachment/primer
sequence 606, such as a P5 attachment sequence, as well as a first
known sequence segment, e.g., a known primer sequence 608, such as
a Read1 primer sequence. Additional functional sequences may
optionally be included, e.g., random primer sequences and the like,
as discussed elsewhere herein, when, for example, a more universal
barcode bead library is used for many different applications or
processes. An additional targeted primer oligonucleotide 610 is
also co-partitioned along with the barcode oligonucleotide 602. The
targeted primer oligonucleotide 610 includes a first portion 612
that provides a complement sequence to a targeted primer sequence,
e.g., a sequence for priming known sequence portion that is
proximal in the sample sequence to a sequence region of interest
(referred to as a targeted primer). As shown, the targeted primer
oligonucleotide 610 also includes a portion, shown as segment 608c,
that is complementary to a portion of the barcode oligonucleotide
602 that is 3' of the barcode segment 604, such as a portion of the
Read1 primer segment 608.
As shown, annealing of the targeted primer oligonucleotide 610 to
the portion of the barcode oligonucleotide 602 and subsequent
extension, e.g., using the polymerase reaction within the
partition, then creates a reverse complement of the barcode
oligonucleotide (shown as 614) with complements of its various
segments (e.g., 604c, 606c and 608c) with the targeted primer
sequence 612 attached, shown as completed oligonucleotide 614.
Further replication of oligonucleotide 614, e.g., using a P5 primer
sequence 616 to prime replication of oligonucleotide 614, e.g.,
that is identical to segment 606 and complementary to segment 606c,
results in the production of a complementary oligonucleotide 618
that includes the barcode segment 620 (that is identical to
barcodes segment 604, as the complement of the complement), the
functional segments, e.g., P5 segment 622 (identical to segment
606) and read1 primer segment 624 (identical to segment 608), and
the targeted primer sequence 626 (complementary to targeted segment
612). The targeted primer sequence 626 is then able to prime
against the targeted portions of a sample nucleic acid 628, that is
also co-partitioned with the barcode oligonucleotides 602 and the
targeted primer oligonucleotides 610, in the same manner described
above for use of the random n-mer primers for generating barcoded
libraries.
As a result, a sequencing library may be created that is
specifically selected for the targeted sequences and which includes
both the barcodes that are indicative of original molecular
context, and one or more desired functional sequences, e.g.,
primers, such as P5, read1, etc.
As will be appreciated, the targeted primer oligonucleotides may be
co-partitioned along with the barcode oligonucleotides by providing
such oligonucleotides in a bulk solution, e.g., and co-partitioning
along with other reagents, e.g., polymerases, dNTPs, etc.
Alternatively, different targeted oligonucleotides or groups of
targeted oligonucleotides may be predisposed on beads similar to
those in the barcode bead libraries described herein, where the
barcode beads and targeted primer beads may be co-partitioned
together into a single partition, e.g., a droplet.
In still a further alternative process, barcoded libraries may be
prepared in a similar fashion to the processes described above, but
through the ligation of the barcode oligonucleotides to the
partitioned fragment nucleic acids. Generally speaking, a fragment
library can be created within a partition from the long fragments
contained within that partition, in order to preserve the molecular
context. The fragment library can be prepared in a fashion that
leaves the fragments available for ligation with the barcoded
oligonucleotides co-partitioned with those fragments, e.g., via a
bead based delivery system as described herein. In certain cases, a
ligation based process can avoid the possibility of amplification
based anomalies, such as priming biases, that could potentially be
associated with an extension based barcoding approach.
One example of such an approach is schematically illustrated in
FIG. 25. As shown, a sample nucleic acid fragment 702 is
partitioned into a droplet or other partition. The long fragment
702 is fragmented into shorter fragments within the partition. As
illustrated, this fragmenting step is carried out by first
replicating the long fragment using a high fidelity polymerase
enzyme, e.g., a phi29 DNA polymerase. The replicating step may be
carried out by priming off of a known terminal sequence segment
that may be provided as an adapter sequence ligated to the
originating fragment, e.g., during a pre-partitioning sample prep
step. Alternatively, and as illustrated, an adapter sequence, e.g.,
adapter sequences 704, may be provided on the originating double
stranded fragment, that provides a known nicking site 706 within
each strand. Following treatment with an appropriate nicking
enzyme, a DNA polymerase capable of priming off of the nicked
strand, e.g., phi29 polymerase, may be used to replicate one strand
while displacing the other strand. This replication can be carried
out with a low level concentration of removable nucleotides, e.g.,
UTP, in order to create a replicate with randomly dispersed uracil
containing bases 708 dispersed throughout its sequence. By using an
enzyme to cleave at the uracil base, e.g., uracil DNA glycosylase
(UDG), e.g., as found in the Uracil Specific Excision Reagent, or
USER (available from New England Biolabs), or other reagents, one
can create a set of fragments of the replicate, e.g., fragments
710, 712, 714, 716 and 718.
Further fragments may be generated by allowing the phi29 polymerase
to extend these fragments from the nicking points, both displacing
the first set of fragments, and creating further replicate copies
that incorporate uracil containing bases at randomly dispersed
intervals, which can then be fragmented as above. Alternatively, a
random priming and extension process, e.g., using random n-mer
primers, e.g., hexamers, 7-mers, 8-mers, 9-mers, 10-mers or larger,
may be used to generate random fragments from the originating
fragment, by annealing to random locations on the originating
fragment, and being extended by a present polymerase, e.g., phi29
or the like. While these alternative priming mechanisms may be
employed, by priming off of random nicking sites, e.g., as
described above, one can reduce priming bias that may come from
exogenously introduced primers, thus allowing creation of a less
biased fragment library from the originating fragment.
Once these fragment libraries are generated, they may be further
replicated using, e.g., random hexamer primers 720 also
co-partitioned with the fragments. The replication of these
fragments using the short primer sequences 720 can result in the
creation of double stranded, blunt ended fragments 722 of varying
lengths. Once the blunt ended fragments 722 are created, they may
be processed in order to attach double stranded barcode
oligonucleotides that are co-partitioned with the fragments, e.g.,
via the bead based delivery systems described herein. For example,
as shown, the blunt ended fragments 722 are first a-tailed, using,
e.g., Klenow polymerase. The A-tailed fragments 724 are then
ligated to the double stranded barcode oligonucleotides 726, e.g.,
including a barcode segment 728, as well as functional sequences,
such as P5 sequence 730 and R1 segment 732, along with the
complementary T base 734 at the ligation point, using a standard
ligation enzyme system, e.g., a T4 ligase. As a result, a barcoded,
double stranded fragment is created. The barcoded fragment may then
be subjected to additional processing as described elsewhere
herein, e.g., to amplify and attach adapter sequences at the other
end.
Additional Processing of Barcoded Libraries
Improvements in library preparation may additionally or
alternatively be achieved through process steps following the
initial barcoding steps, described above. For example, following
the creation of barcoded replicate fragments of the template
nucleic acid, e.g., as described above, additional processing may
be carried out with the barcoded fragments, e.g., to further
amplify those fragments and/or to provide additional functional
sequences on those fragments or copies thereof, e.g., additional
sequencing primers, sample index sequences and the like.
In many cases, the barcoded replicate fragments may be further
processed to both provide greater quantities of barcoded nucleic
acids for sequencing, and also to attach additional functional
nucleic acid sequence segments to the library members in order to
efficiently process the library on a sequencing system. Because
this additional processing occurs after the attachment of the
barcode sequences to the fragments, e.g., preserving the linkage
information of fragments generated from a given nucleic acid
molecule within a given partition by virtue of the common included
barcode sequences, the subsequent processing may be carried out as
a pooled reaction, e.g., where the contents of the various
partitions are pooled together for bulk processing.
By way of example, as described in U.S. patent application Ser. No.
14/316,383, filed Jun. 26, 2014, and previously incorporated herein
by reference, the barcoded fragment nucleic acids, e.g., fragment
126 in FIG. 19E, can be subjected to additional processing to
amplify the presence of those fragments, as well as to attach
additional functional sequences for use in sequencing processes.
For example, once the barcoded fragments 126 are prepared within
individual partitions, the various separate partitions may be
ruptured (e.g., by breaking the aqueous in oil emulsion), resulting
in a pooling of all of the barcoded fragments that originated from
different partitions and bearing different barcode sequences. The
amplification of the barcoded fragment 126 may then be carried out
by priming against the replicated functional sequence, e.g., the R1
complementary sequence 114', where the primer for this
amplification also includes additional functional sequences, e.g.,
the P7 and R2 sequences, or their complements. As a result, the
produced sequences can include on each end the requisite functional
sequences or their complements. Further, one may amplify by
anteparallel priming by also using a primer against the original
functional sequence 110, as the primer annealing sequence, to
initiate anteparallel amplification, e.g., PCR.
One exemplary process is illustrated in FIG. 22, and with reference
to FIGS. 19A-F. In particular, assuming a barcoding process as
shown in FIGS. 19A-F, one could obtain a barcoded set of nucleic
acid fragments 402 in FIG. 22, that would be a pooled set of
fragments, e.g., from multiple partitions, and bearing multiple
different barcode sequences on the attached barcode oligonucleotide
408, including the barcode sequence(s) 412 along with the other
functional sequences, e.g. attachment sequence 410 and sequencing
primer 414, attached to the sample fragment or insert 422.
A second set of primer sequences 450 would then be introduced into
the reaction mixture. As shown, the second set of primer sequences
450 includes additional functional sequences used in sequence
libraries, e.g., for attachment to sequencer flow cells, e.g., the
P7 sequence 452, and for priming of the second reading step for the
sequencer, e.g., R2 priming sequence 454. Also included in these
primer sequences could be a set of random priming sequences, e.g.,
random n-mer 456, as well as optional sample index sequences (not
shown), that would be common for any given sample. The random n-mer
456 c randomly prime against the barcoded fragments 402 in the
reaction mixture, and extension of these primers would produce a
replicate copy 458 of the barcoded fragment 402, including a
complementary replicate of the barcode oligonucleotide 408, e.g.,
including a complement to barcode sequence 412 (shown as segment
412c) and complements to any functional sequences included in that
barcoded fragment, e.g., P5 attachment sequence 410 (shown as
complementary sequence 410c) and R1 primer sequence 414 (shown as
complementary sequence 414c).
Following this replication, the resulting fragments 458, now
including functional sequences at both ends, e.g., the P5 and P7
sequences (segments 410 and 452, respectively) of an insert
sequence segment 460. These completed fragments may then be
subjected to additional amplification steps, e.g., PCR, using the
known terminal segments of the fragments, e.g., the P5 and P7
sequences or their respective complements such as segments 410c and
452, as priming regions for anteparallel amplification.
As will be appreciated, in some cases following the initial
generation of barcoded fragments, it may be desirable to purify the
barcoded fragments away from the reaction mixture that was used to
produce them, e.g., using SPRI beads, etc. For example, when using
a polymerase that is incapable of processing through uracil
containing bases, e.g., as described above with reference to FIGS.
19A-F, it may be useful to swap out that polymerase for a different
polymerase to be used to further process the fragments, allowing
replication of the uracil containing portion of the barcode
oligonucleotides as shown in FIG. 22. A variety of different,
highly processive, highly accurate polymerases may be employed in
this process, including for example, thermally stable polymerases,
e.g., taq, 9.degree. North, Deep Vent polymerases, as well as
non-thermally stable polymerases, e.g., Bst, Klenow, phi29, and the
like. In some cases, e.g., as described above for hairpin or
partial hairpin structures, it may also be desirable to utilize
polymerases in the subsequent amplification steps that possess one
or more of strand displacing activity, uracil tolerance, proof
reading capability, e.g., including exonuclease activity, and the
like.
Likewise, following the second replication step, e.g., as
illustrated in FIG. 22, it may be desirable to purify the
replicated fragments 458 prior to subjecting them to further PCR or
other amplification in order to remove extraneous primers
sequences, e.g., primers 450, from participating in the selected
amplification of the resultant fragments 458.
Although illustrated as incorporating the functional sequence
segments in the primer set 450, it will be appreciated that some of
these sequences may be incorporated in subsequent process steps.
For example, in some cases, the primer set 450 might not include a
functional sequence like a P7 sequence, e.g., segment 452.
Following replication of the barcoded fragments, one can add
additional functional sequences to the resulting library of
fragments, e.g., fragment 458. Again, addition of other sequences
can be accomplished through a ligation step, e.g., as described
below with reference to FIG. 23, or alternatively, it could be
introduced as a component of a primer sequence used in a subsequent
amplification of the resulting fragment 458. In particular, an
additional sequence could be provided attached to a primer sequence
that can prime against a portion of the fragment, e.g., segment 454
(assuming the absence of fragment 452). Amplification of the
fragment 458 can then carry with it the sequence segment added
through the primer.
In another exemplary process, subsequent processing of the initial
barcoded fragments, e.g., fragment 118 or 402 from FIGS. 19A-F or
22, respectively, can be achieved through a shearing and ligation
process to provide finished fragments bearing the requisite
functional sequences. This process is schematically illustrated in
FIG. 23. As shown, a collection of barcoded, double stranded
nucleic acid fragments 502 is produced from the initial barcoding
step, e.g., as shown in any of FIGS. 19 and 20. The fragments and
their associated complementary strands 504, e.g., the templates
from which they were replicated, are then subjected to a shearing
process, e.g., using enzymatic, mechanical and/or acoustic shearing
processes, e.g., Covaris AFA shearing processes, to produce sheared
double stranded fragments 506.
The sheared double stranded fragments 506 are then blunt ended
using, e.g., one or more of fill-in reactions, e.g., using Klenow,
and/or nuclease treatments. Following blunting, an A base is added
to the 3' terminus, e.g., using a Taq or other non-proofreading
polymerase in the presence of dATP, to yield the A-tailed, blunt
ended double stranded fragments 508. Adapter 550, which includes a
T-base at its 3' terminus, is then added to the mix in the presence
of appropriate ligation mixture, e.g., T-4 ligases and associated
reagents. As shown, the adapter 550 includes the additional
functional sequences needed for application to the sequencer of
choice, e.g., the Read2 primer (complement) 552 and P7 (complement)
554 sequences. Also included is a partially complementary sequence
having a 3' T-base overhang (shown as partial R2 segment 556), in
order to allow efficient ligation with the barcoded fragments.
Following ligation, the resulting library element 558 includes the
insert sequence 560, e.g., derived from the original sample
template sequence, the first set of functional sequences, e.g., P5
510 and R1 514 sequences, the barcode sequence 512, and a second
set of functional sequences, e.g., R2 and P7 sequences or their
complements (segments 552 and 554, respectively). Also included are
the original primer sequences 516 from the barcoding
oligonucleotides.
As described above, the resulting barcoded fragments may then be
further amplified by priming amplification, e.g., anteparallel
amplification like PCR, using the known end sequence segments,
e.g., P5 sequence 510 and P7 sequence 554, as the priming
targets.
As will be appreciated, in some cases, the shearing step described
above can produce fragments where the original barcoded sequence
has been sheared off, or can produce fragments that result from
sheared fragments that did not include the barcode fragments.
Because these fragments lack a complete set of functional
sequences, e.g., both of P5 and P7, or any other functional
sequences used to prime subsequent amplification steps, even
following ligation of the second set of functional sequences, e.g.,
through adapter 550, they would not be amplified in subsequent
steps, which rely on the presence of both sets of sequences, e.g.,
P5 and P7 sequences, for successful amplification. Restated,
although incorrectly ligated fragments may initially be created,
they may not be subsequently amplified and, as a result, can fall
below the noise level of the system upon sequencing.
A number of additional or alternative processes may be employed in
further processing the barcode library elements. For example, when
starting with barcoded nucleic acid fragments, e.g., fragment 126
in FIGS. 19E-F, or other similar barcoded fragments, one may attach
additional functional sequences to the end of the fragment, e.g.,
the non-barcoded end, via a number of methods. For example, as
noted above, this may be achieved through the amplification of the
total sequence from the non-barcoded end using a primer that
includes additional functional sequences, such that the extension
products of such primer include not only a copy of the barcoded
fragment 126, but also the functional sequences attached to the
primer. Likewise, additional sequences may be simply ligated to the
end of the sequence to add functional sequences.
A number of other process steps may be employed in further
processing, amplifying, and/or appending additional sequences to
the barcoded fragments described herein. For example, in some
cases, rather than creating a partial hairpin, e.g., using uracil
containing bases in the barcode oligonucleotides to block complete
replication, non-uracil containing barcode oligonucleotides may be
used to permit formation of complete hairpin molecules. By
selectively removing a portion of the 3' terminus, one may create a
ligation site for the additional functional sequences for the
various fragments. For example, by incorporating the complement to
a nicking enzyme recognition site in a common known portion of the
barcode oligonucleotide, e.g., in the R1 primer segment, described
above, one could indirectly create a nicking site in the downstream
portion of the hairpin duplex. Treatment of the hairpin with the
requisite nicking enzyme could yield a partial hairpin structure
having a portion of single stranded DNA that is known, which known
sequence portion may be used as a landing spot for ligation of an
additional functional sequence(s) to the 3' end of the partial
hairpin, e.g., read2, P7, sample indices, etc.
In an alternative, but related approach, one may create a complete
hairpin structure using the approach outlined in FIGS. 19A-F, but
employing a polymerase enzyme that is capable of processing through
uracil containing bases. In such case, a fragment that results from
initial extension of a barcode containing primer oligonucleotide,
e.g., uracil containing oligonucleotide 108, is completely
replicated through the extension of a second barcode containing
primer oligonucleotide, e.g., oligonucleotide 108b, such that the
complete replicate includes barcode oligonucleotide 108b (including
the uracil bases) at one end, and a complement of the original
barcode oligonucleotide 108 (without uracil containing bases) at
the other, which would include a complement to the barcode segment
112, and the functional sequences, e.g., 110 and 114. One could
then cleave the resulting replicate fragment at the uracil
containing bases, e.g., using a UDG enzyme or the like, to leave a
portion of the barcode oligonucleotide 108b on the end of the
fragment, e.g., segment 114. The other end, meanwhile, can still
retain the complement to the original barcode oligonucleotide,
including the complement to the barcode sequence and functional
sequences. By leaving a known segment attached to the digested end,
one is provided with a handle at which to ligate the second side
adapter sequence, e.g., including other functional sequences, e.g.,
sequencer specific attachment and primer sequences, sample index
sequences, and the like.
In still other aspects, one may exploit the hairpin structure of
the barcoded fragments created in a barcoding process. For example,
in some cases, it may be desirable to create a barcoded fragment
that forms into a complete hairpin structure, as noted above. With
reference to the process described above and shown in FIGS. 19A-F,
for example, one could provide complete barcoded hairpin structures
by allowing complete replication of the barcode/primer sequence,
with or without additional functional sequences included. The
termini of the duplexed portion of the hairpin may then be treated
as a terminus of a standard duplex in duplex adapter attachment
process (see, e.g., Illumina Truseq Sample Preparation Guide
(Illumina, Inc. part #15026486 Rev C), and U.S. Pat. No.
8,053,192), the full disclosures of which are incorporated herein
by reference in their entirety for all purposes), to attach the
additional functional sequences to the hairpin. In particular, the
Truseq adapter includes both the P5-Read2 sequence in a partial
hybrid structure with the P7-Read2 sequence, based upon at least
partial complementarity between the read1 and read2 primer
sequences. As a result, the duplex portion of the adapter may be
attached, e.g., ligated, to the duplex end of the hairpin
structure, to attach the P5-Read 1 sequence to the 5' end of the
hairpin molecule, and P7-R2 to the 3' end of the hairpin. As
described above, once the duplex adapter is attached to the duplex
end of the hairpin, it may be amplified, e.g., using an
ante-parallel, PCR amplification process by priming against the P5
and P7 sequences. As will be appreciated, one could attach a
variety of different additional functional and other sequences to
the ends of the hairpin structure using partial or completely
complementary and duplexed structures that are ligated to the
hairpin, using this approach.
Alternative processes may likewise be used to modify complete
hairpins that include the barcode oligonucleotide structure. In
particular, rather than generating partial hairpin structures, one
could incorporate a selective nicking site into the complementary
duplex structure that allows nicking of the 5' portion of the
duplex, which when digested, can yield a partial hairpin structure,
which may then be processed as discussed above.
Additional Systems and Kits
Although primarily described in terms of the library generation and
preparation processes, it will be appreciated that also provided
herein are process systems, reagents, consumables and reagent and
consumable kits used for carrying out the above-described
processes. For example, overall systems may include the reagents
necessary for carrying out the above-described reaction processes,
e.g., including barcoding reagents such as barcode oligonucleotide
libraries disposed on partitionable beads, e.g., as described in
detail in, for example, co-pending U.S. patent application Ser. No.
14/316,383, filed Jun. 26, 2014, 62/017,808, filed Jun. 26, 2014,
62/072,214, filed Oct. 29, 2014, 62/072, filed Oct. 29, 2014, and
62/017,558, filed Jun. 26, 2014, previously incorporated herein by
reference in their entireties for all purposes. Also included in
such systems may be other reagents used in the process, such as
partitioning fluids, e.g., fluorinated oils, nucleoside
triphosphates, and the like, as well as partitioning systems used
to co-partition sample nucleic acids with the barcode reagents,
including both microfluidic consumable components in which
partitions are generated as well as instruments used to drive and
control the operation of the microfluidic devices.
As noted, kits are also provided herein that include the reagents
necessary for carrying out the reaction processes described herein.
Typically such kits can include the barcoding reagents including
the requisite barcode oligonucleotide bearing bead libraries, and
appropriate enzymatic reaction reagents, e.g., appropriate
polymerase enzymes, monomers, and other reagents, e.g., UDG, USER
or the like, for carrying out the desired reaction. The kits
likewise may also contain the requisite partitioning reagents, such
as the non-aqueous partitioning fluids, e.g., fluorinated oils, and
the like. Finally, the kits can also typically include user
instructions for directing the user to carry out the desired
reaction process as described in detail above.
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one
skilled in the art from a reading of this disclosure that various
changes in form and detail can be made without departing from the
true scope of the invention. For example, all the techniques and
apparatus described above can be used in various combinations. For
example, particle delivery can be practiced with array well sizing
methods as described. All publications, patents, patent
applications, and/or other documents cited in this application are
incorporated by reference in their entirety for all purposes to the
same extent as if each individual publication, patent, patent
application, and/or other document were individually and separately
indicated to be incorporated by reference for all purposes.
X. EXAMPLES
Example 1: Molecular Barcoding of Priming Free Amplification
Templates
It is contemplated that a number of approaches would be effective
for molecular barcoding templates resulting from priming free
amplification for sequencing. The reactions and reagents for
achieving molecular barcoding can be part of the same reaction and
run simultaneously with the priming free amplification of
templates. The approach can include adaptors as well. For example
adaptor designs can include partial R1 sequence from Illumina's
primer design, followed by a preferred barcode sequence followed by
a random Nmer (sequence size varies between 2-20 bases). These
adaptors can be double stranded and include a barcode and R1
sequence with the Nmer arranged as a 3' overhang.
In a first approach, as shown in FIG. 2A, barcoding the templates
can be achieved using an extension barcoding approach. Stand
displacement and high processivity of phi29 DNA polymerase releases
amplified fragments thereby enabling recycling of the template for
further amplification. The single strand fragments that are
generated during stand displacement can be converted to dsDNA but
the hexamer or Nmer part of the adaptor by the same polymerase.
Another approach to molecular barcoding is shown in FIG. 2B.
Amplified templates generated as described in FIG. 1 are molecular
barcoded optionally by a single stranded or double stranded
template to barcode ligation approach. As shown, the template DNA
molecules are converted to either single stranded (using
temperature/enzyme; see left half of figure) or double stranded
(using enzyme; see right half of figure). The molecular barcodes,
e.g., oligonucleotides are attached through a ligation process
using a ssDNA ligase (ovals) or dsDNA ligase (ovals) or other
nucleic acid modifying enzymes. Additional oligonucleotides serving
as molecular handles may be added to the first barcode tag in
subsequent ligations.
An additional approach to molecular barcoding the templates is
shown in FIG. 2C. In this scheme, a single strand DNA molecule
(with barcode/primer sequence) is attached to the bead from 3' end.
The 5' end of the oligo is pre-adenylated (either chemically or
enzymatically). The oligo can be sequestered using Hotstart-IT
binding protein if desired which can be released using heat. For
barcoding the single-stranded library molecules (single strands
generated by heat treatment or helicase), APP DNA/RNA ligase will
ligate 5' pre-adenylated oligo with 3' end of the library molecule.
This process is very specific as oligo-oligo ligation can be
avoided by blocking the 3' end and library molecules cannot self
ligate as they are not adenlyated.
APP DNA/RNA ligase can be a thermostable 5' App DNA/RNA Ligase
including a point mutant of catalytic lysine of RNA ligase from
Methanobacterium thermoautotrophicum. This enzyme is ATP
independent. It requires a 5' pre-adenylated linker for ligation to
the 3'-OH end of either RNA or single stranded DNA (ssDNA).
A further approach to molecular barcoding the templates uses a
topoisomerase enzyme. For example, topoisomerase I from Vaccinia
virus binds to duplex DNA at specific sites and cleaves the
phosphodiester backbone after 5'-CCCTT in one strand. Here
molecular barcoding can be achieved where at an adapter sequence
(e.g., an oligonucleotide) is pre-bound to a topoisomerase enzyme.
The amplified templates can be prepared for blunt end ligation
using, for example, the Klenow fragment of DNA polymerase.
Example 2: Priming Free Amplification by Polymerization at Nick
Sites Results in Thymidine (T) Base Bias
Experiments were conducted using an amplification protocol with (A)
or without primer (B).
(A) Amplification Protocol with Primer Formulation:
1.times. Thermopol Buffer (NEB), 0.2 mM dNTP Mix (10 mM each), 0.3
uM Primer*, 0.07% (v/v) Glycerol, 0.5% (w/v) Synperonic-F108, 1 mM
DTT, 0.1 ng/.mu.L gDNA Template, 0.4 U/.mu.L 9.degree. N
Polymerase.
Primer Seq:
TABLE-US-00001 (SEQ ID NO: 1)
TAGAUCGCACACUCUUUCCCUACACGACGCUCTTCCGATCNNNNNNNNNN
Thermocycling Protocol:
1.) 4.degree. C./.infin.
2.) 98.degree. C./5:00 mins--ramp 2.degree. C./S
3.) 4.degree. C./0:30 sec--ramp 2.degree. C./S
4.) 45.degree. C./0:01 sec--ramp 0.1.degree. C./s
5.) 70.degree. C./0:20 sec--ramp 2.degree. C./S
6.) 98.degree. C./0:30 sec--ramp 2.degree. C./S
7.) go to Step 2, 14.times.
8.) 4.degree. C./.infin.
(B) Amplification Protocol without Primer (Priming Free
Amplification by Polymerization) Formulation:
50 mM Tris, pH 7.5, 10 mM (NH4)2SO4, 0.50% SymPeronic, 1 mM dNTP,
0.03 mM dUTP, 7% Glycerol, 25 uM Hexamer, 17 mM DTT, 1 ng gDNA, 10
ug/ml BSA, 0.01% Triton X, 0.006 U/ul UDG, 30 U/ul EndoIV, 0.2 uM
Phi29 DNA Pol
Thermocycling Protocol:
1.) 30.degree. C./3 hours
2.) 65.degree. C./10:00 mins
3.) 4.degree. C./.infin.
Using a priming free amplification by polymerization reaction,
dUTP's (U) were incorporated into templates. Excision of "U" was
achieved with a lyase enzyme creating a nick in the template which
resulted in an initiation site for the polymerase. Since the
initiation occurred as a result of the U excision, there is a bias
for the base Thymidine (T) that's reflected in the sequences
observed.
As shown in FIG. 3, testing for T base bias based on whole genome
sequencing data revealed a bias for T base. The T base bias scaled
proportionately with dUTP concentration tested, strongly supporting
that most initiation was driven by U incorporation/excision. The T
base bias was revealed when the sequences were aligned to a
reference sequence.
The results shown in FIG. 3 validated the concept of polymerase
initiation from the created nick sites rather than the primer based
extensions.
Example 3: GC Coverage: Primed Amplification Vs. Priming Free
Amplification
The two plots in FIGS. 4A and 4B show coverage evenness over 1000
bp binned GC content of the human genome. As can be seen from the
plots, the primed amplification reaction (FIG. 4A) does not have
even coverage whereby the low GC and high GC genome regions are
poorly represented as compared to regions with GC content of
0.35-0.5. In comparison, the primer free amplification method (FIG.
4B) shows even coverage across broad range of GC contents.
Example 4: Titration of dUTP for Effect on GC Coverage
GC coverage plots illustrated in FIGS. 5A-5E shows the evenness of
coverage using sequencing across different parts of the genome
binned by their GC content. The data shows that GC coverage is more
skewed towards high GC when there is no dUTP present (FIG. 5A), and
it becomes more even with higher dUTP (>1%). Results for no
dUTP, 0.5%, 1%, 2% and 3% dUTP are shown in FIGS. 5A, 5B, 5C, 5D
and 5E respectively. In sum, it was observed that use of >1%
dUTP, when compared to no dUTP (FIG. 5A) or 0.5% dUTP (FIG. 5B),
advantageously results in even coverage of various GC bins.
Example 5: Titration of dUTP for Chimera Reduction
In a priming free amplification by polymerization reaction, dUTP
concentration during amplification was titrated and the effect on
chimera rate from reads in the same direction, Depth Positional
Coefficient of Variation (DPCV) deduped on confident regions and
amplification (amp) rate from full coverage over 1000 bases were
studied. As shown in FIG. 6, in a range from about 3.5% to about
5.5% dUTP, significant reduction in chimera rate was observed while
both DPCV and amp rate remained relatively strong and stable.
Example 6: Addition of DTT Reduces DPCV
DTT addition was tested for the effect on DPCV and amplification
rate in priming free amplification by polymerization reactions. As
shown in FIG. 7, addition of DTT was tested over a concentration
range of 1.0 mM to 10 mM. Advantageously, across the range of
tested DTT concentrations, beneficial reduction in DPCV was
observed without appreciable effect on the amplification rate.
Higher concentrations of DTT resulted in even more reduction in
DPCV. As such DPCV was improved with the addition of DTT without
adversely affecting amplification rate.
Example 7: Polymerization Conditions Optimization for Whole Genome
Analysis
Various reaction components for priming free amplification by
polymerization reactions were tested in a number of combinations to
determine optimized polymerization for whole genome template
sample. As shown in FIG. 8A, the standard condition including
addition of SSB, DTT or both had lower DPCV as compared to similar
condition with higher dUTP (5%) concentration. As shown in FIG. 8B,
the data suggested that addition of SSB reduced amplification rate,
which was reduced even further in presence of 5% dUTP. As shown in
FIG. 8C SSB reduces chimeras as compared to conditions where SSB
was omitted. DTT also reduced amp rate.
Example 8: Polymerization Reaction Time Course
In a bulk priming free amplification by polymerization reaction
using phi29 polymerase at 32 nM, both DPCV and amplification rate
were measured over time, up to 8 hours. As shown in FIG. 9, the
DPCV improves (is reduced) slightly from 1 to 4 hours (0.22 to
0.20) and essentially plateaus over the remaining 4 hours. The
amplification rate (shown as BAC-aware Amp) remained relatively
flat across the entire time series tested. Additional phi29,
testing at 80 nM did not significantly impact the above results
(data not shown).
Example 9: Effect of Template Denaturation on DPCV and
Amplification Rate
To test the effect of template denaturation on DPCV and
amplification rate in priming free amplification by polymerization
reactions, three conditions where tested in blank GEMs: i) no
denaturation (no heat), ii) NaOH denaturation and iii) heat
denaturation. Experiments were performed in duplicate. As shown in
FIG. 10, the results of the experiment indicated that DPCV is
fairly stable in all conditions tested but amplification is
substantially lower when the template is not denatured. As tested,
either NaOH or heat denaturation can effectively be used for
successful polymerization. However, a slight advantage for heat
denaturation was observed.
Example 10: Titration of Adaptor Concentration
The suitable range for adaptor concentration for molecular
barcoding was tested by titration of and adaptor and measuring DPCV
and dup rate. The tested conditions were 0.4 U/uL Phi29 DNA
polymerase, 54 nM-500 nM adaptor 12 (duplex pR1 in-line BC
adaptor).
As shown in FIG. 11, both DPCV and dup rate was stable between the
tested range of 54 nM-500 nM adaptor, although an increase in
unmapped fraction was observed as adaptor concentration
increased.
It is expected from these results that the suitable range of
adaptor concentration might be extendable to 1 nM-10 uM by
including SSB (single stranded binding protein) or other additives
to reduce the unmapped fraction.
The table in FIG. 11 shows the effect of adaptor concentration on
dup rate (measure of library complexity) and DPCV (measure of
coverage evenness). The first column shows the adaptor
concentration used with `LL ctrl` sample has no adaptors. The third
column shows the depth of sequencing (deduped--duplicates are
removed before calculating this number). The fourth column shows
the dup rate post downsampling all the samples to 0.25.times.
coverage, this number is also calculated using the barcode
information. The fifth column shows DPCV, measure of coverage
evenness. The results shown indicated that across a broad range of
adaptor concentrations, the dup rate and DPCV remains relatively
flat suggesting the reaction's tolerance to broad range of adaptor
concentrations.
Example 11: Effect of Barcoding Ligation Reaction Time
This experiment was designed to study the effect of reaction
duration on different sequencing matrices. The study was conducted
at two different adaptor (adptr) concentrations, 0.2 uM and 2
uM.
FIG. 12A shows: DPCV reduces with shorter reaction time; FIG. 12B
shows: insert size increases with shorter reaction time; FIG. 12C
shows: chimeras are reduced with shorter reaction time; FIG. 12D
shows unmapped fraction is unaffected as a function of time; and
FIG. 12E shows: at lower adaptor concentration, the amplification
(Amp) rate is flat, higher adaptor concentration shows increase in
amplification after 4 hours. Based on these results, 3 hours of
reaction time can be interpreted to be optimum of most
matrices.
Example 12: T4 Ligase Molecular Barcoding of Priming Free
Amplification Products
FIG. 13 shows the results of control experiments to test the
specificity of T4 ligase based barcoding. The readout is P5/P7
quants. P5/P7 quant of >5 is considered positive. The results
show that it is necessary to have ligase, template, and adaptor
present to make a useful set of barcoded templates (e.g., a library
of templates for sequencing). Absence of any of the three
components results in an inadequate set of barcoded templates for
use, for example as a library of amplified templates for
sequencing.
Example 13: Evenness of Sequencing Coverage--Primed Amplification
Vs. Priming Free Amplification
FIGS. 14A and 14B are histograms comparing the coverage evenness
between primed amplification (FIG. 14A) and priming free
amplification (FIG. 14B). The y-axis in both figures is the number
of genomic locations. The x-axis plots increasing coverage from
left (0) to the right. The data clearly shows the improved coverage
eveness advantage observed in the priming free amplification
protocol, which had a more poissonian distribution when compared to
the distribution for primed amplification.
Example 14: Concentration of nMer (uM) Effect on DPCV
The effect of nMer concentration (uM) was tested on five different
barcoded template library samples prepared as described above. As
shown in FIG. 15, at higher concentrations of nMer, above 30 uM,
advantageously reduced DPCV in four out of five samples was
observed. At 40 uM and 50 uM, every sample showed reduced DPCV with
the greatest reduction being observed at 50 uM nMer concentration.
The results indicated that higher rather than lower concentrations
of nMer are required for improved DPCV reduction.
Example 15: SPRI Stringency Cut Effect on DPCV
The effect of SPRI (Solid Phase Reversible Immobilization)
stringency cut was tested on six different barcoded template
library samples as described above. As shown in FIG. 16, more
stringent SPRI cuts advantageously resulted in reduced DPCV.
Example 16: Total Reaction Time Effect on DPCV
The effect of total reaction time on DPCV was tested on five
different barcoded template library samples as described above. As
shown in FIG. 17, under the instant test conditions, the DPCV is
relatively unaffected by time. Time points tested ranged from 2
hours to over 10 hours.
Example 17: USER Concentration Effect on DPCV
The effect of USER.TM. (Uracil-Specific Excision Reagent; New
England Biolabs.RTM. Inc. (NEB), Ipswich, Mass.) concentration on
DPCV was tested on six different barcoded template library samples
as described above. As shown in FIG. 18, under the experimental
test conditions, on average the DPCV is relatively unaffected by
USER concentration.
It should be understood from the foregoing that, while particular
implementations have been illustrated and described, various
modifications may be made thereto and are contemplated herein. It
is also not intended that the invention be limited by the specific
examples provided within the specification. While the invention has
been described with reference to the aforementioned specification,
the descriptions and illustrations of the preferable embodiments
herein are not meant to be construed in a limiting sense.
Furthermore, it shall be understood that all aspects of the
invention are not limited to the specific depictions,
configurations or relative proportions set forth herein which
depend upon a variety of conditions and variables. Various
modifications in form and detail of the embodiments of the
invention will be apparent to a person skilled in the art. It is
therefore contemplated that the invention shall also cover any such
modifications, variations and equivalents. It is intended that the
following claims define the scope of the invention and that methods
and structures within the scope of these claims and their
equivalents be covered thereby.
SEQUENCE LISTINGS
1
1151DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primerDescription of Combined DNA/RNA Molecule Synthetic
primermodified_base(42)..(51)a, c, t, g, u, unknown or other
1tagaucgcac acucuuuccc uacacgacgc ucttccgatc tnnnnnnnnn n 51
* * * * *
References